Part Seventh
THE ORIGIN AND ESTIMATION OF OXIDIZED NITROGEN IN SOILS, RAIN AND DRAINAGE WATERS.
=411. Introductory Considerations.=—The estimation of oxidized nitrogen in the soil would properly find a place in the preceding part; but on account of the late progress in our knowledge of the source of this indispensable and costly plant food it has become necessary to give it especial attention. The present part will, therefore, be devoted to a brief statement of our present knowledge in respect of the origin of oxidized nitrogen, a description of the nitrifying ferments and methods for their isolation and determination and finally the most approved methods of estimating the ammonia, nitrous, and nitric acids formed thereby both in the soil and the waters pertaining thereto or proceeding therefrom. It is scarcely necessary to caution the reader not to consider this part in any sense a treatise on the bacteria active in soil chemistry. Its object is rather to place in the hands of the soil analyst data which will enable him to intelligently study the soil phenomena depending on these organisms and to determine the extent and character of their biological and chemical functions. These are matters which, up to the present time, have found no place in manuals dedicated to agricultural analysis.
=412. Organic Nitrogen in the Soil.=—With the exception of the small quantities of nitric acid added to the soil directly by rain water, the whole of the supply of this substance is derived from the products of the oxidation of nitrogenous bodies. These products are either stored as the results of past nitrification or are formed synchronously with their consumption by the growing plant. Nitrogenous compounds are present as organic vegetable or animal remains and as humus. All vegetable and animal material deposited in or on the soil contains more or less of these proteid or nitrogenous matters while the amount of nitric acid supplied in this way is probably represented entirely by the quantity in the organism of the plant or animal and unabsorbed at the time of its death. In other words it is not demonstrated that nitrates or nitrites are in any sense a special product of plant growth save in the case of nitrifying organisms themselves which are supposed to be of a vegetable nature. Animal organisms do not in any sense assimilate nitric nitrogen.
With most plants, the quantity of proteid nitrogen which they can deliver to the soil is in no case greater than the sum of organic and nitric nitrogen supplied in their food and they can therefore be regarded only as the carriers and conservers of this substance. On the other hand there are some plants notably those belonging to the leguminous family which permit of the development on their rootlets of colonies of bacteria which have the faculty of rendering atmospheric nitrogen available for plant growth. Whether or not there exist plants other than the micro-organisms mentioned which are capable of directly oxidizing and fixing atmospheric nitrogen is still an unanswered question. It is not probable, however, that the difficult task of oxidizing atmospheric or free nitrogen would be accomplished in nature in only one way. In fact it has already been established that organisms do exist which are capable of oxidizing free nitrogen in a manner wholly independent of other plant life and to produce weighable quantities of nitric acid when developed in media of mineral matters and pure carbohydrates to which free nitrogen has access. It is, therefore, fair to assume that the fixation of free nitrogen is a function of chemical activity quite independent of ordinary plant life and that the leguminous plants take no further part in this process than that of providing in their radical development a favorable nidus for the growth of the nitrifying organism.
By the action of denitrifying organisms a portion of the nitrogen of nitric acid is constantly restored to a free state, a far larger portion, perhaps, than is fixed in the atmosphere itself by the action of electricity. Were it not, therefore, for the activity of the nitrifying ferments the stores of nitrogen available for growing plants would constantly become less. Instead of this being the case, however, it is probable that the contrary is true and that, by a wise system of agriculture, the total nitrogen at the disposal of plants may become greater and greater in quantity.
=413. Development of Nitric and Nitrous Acids in Soils.=—Owing to the solubility of nitrates there can be but little accumulation of them in soils in those countries where there is any considerable amount of rain-fall. On the other hand in arid regions there may be found extensive deposits of nitrates. The occurrence of a certain quantity of nitrates in the soil, however, is essential to the growth of plants. Until within a few years little was known of the origin of nitric acid in the soil. The presence of nitrates in drainage waters was well established, likewise the consumption of nitric acid by the growing plant, but the method of its supply was unknown. In a general way it was said that the nitric acid came from electrical action and the oxidation of the albuminous bodies in the soil, but without specifying the manner in which this change takes place. The researches of Schloesing and Müntz, of Springer, Winogradsky, Frankland, Warington and others have demonstrated the fact that this oxidation is caused by means of bacteria and that the nitrates formed can be consumed and destroyed by other species of this organism. In the one case the process has been called nitrification and in the other denitrification.[274]
The influence of these low organisms both in producing fertility in a soil and maintaining it in a state of fertility is of the highest importance.
=414. Conditions Necessary for Nitrification.=—In order to properly understand the reasons for many of the steps in investigating a soil for nitrifying organisms, it will be useful to state the general conditions on which nitrification depends.
The nitrifying organism, like every other one, first of all feels the necessity for food. In general, food which is given to microbes of all kinds consists of some organic matter together with the addition of mineral substances necessary to growth. These substances in general are phosphoric acid, potash, and lime. Of these articles of bacterial food phosphoric acid seems to be the most important. With the nitrifying organisms, however, it has been found that the organic matter can be omitted. In fact, as will be seen further on, the omission of organic matter supplies the best condition for the proper isolation of the organisms. In other words some forms of the nitrifying organisms have the property of subsisting wholly on mineral substances, i. e., are true vegetables.
The presence of oxygen is also necessary to the growth of the common nitro-organisms. In an atmosphere deprived of oxygen or in which the oxygen is reduced to a very low percentage, the process of nitrification is retarded or stopped as the oxygen diminishes or disappears.
The presence of a base with which the nitrous or nitric acid formed may unite is also essential to the proper conduct of the process. For this reason the nitrification should take place in a solution which is feebly alkaline or in the presence of a base which can be easily decomposed so that no acidity can take place. Calcium carbonate is a base well suited to favor the nitrifying process and its presence in a soil favors the rapid oxidation of proteid matter. The mistake must not be made, however, of supposing that an excess of alkali would favor nitrification. The contrary is true. A slight excess of alkali may prevent nitrification altogether when it is due to the common organisms present in an arable soil. It may be that in soils charged with alkali a different organism exists which is capable of exercising its functions when the alkali is in excess.
The temperature to which the nitrifying body is subjected is also a matter of importance. The nitrifying organisms have the property of remaining active at lower temperatures than most bodies of their class. On the contrary their action is retarded and destroyed by high temperatures. The most favorable temperature for nitrification is about that of blood heat; viz., 37°. At 50° the organism shows very little activity and at 55° its activity ceases altogether. Nitrification, however, according to Warington, cannot be started in a solution if the initial temperature is 40°.
Desiccation has the same retarding influence on nitrification that a high temperature has. Even thoroughly air-drying a soil may destroy its nitrifying qualities.
Darkness is also necessary to the proper progress of nitrification. In a strong light, the activity of the organism is very much diminished or destroyed altogether. A bright light like sunshine may even stop nitrification which has set in.
=415. Effect of Potassium Salts on Rate of Nitrification.=—Dumont and Crochetelle have described some experiments to determine the effect of potassium salts alone and in combination with lime on nitrification.[275]
Soil rich in vegetable mold (18.5 per cent of humus and 0.29 per cent of lime) was treated with varying amounts of potassium sulfate and carbonate and kept for twenty days at 25°. In the untreated soil the amount of nitric acid produced was twenty-five parts per million. When potassium carbonate was applied in quantities of from one-tenth to six per cent the amount of nitric acid increased from forty-seven parts per million to 438 parts when four and one-half per cent of the potassium salt were used. Larger quantities caused a decrease in the amount of nitric acid produced. Very little effect, on the contrary, was produced by the action of potassium sulfate. When one-half per cent was employed the quantity of nitric acid formed rose to fifty parts per million, while with quantities as high as five per cent it fell below the normal; viz., twenty-five parts per million.
When calcium carbonate was added to the soil in conjunction with potassium sulfate there was a marked increase in the amount of nitrogen oxidized. The activity of potassium sulfate in promoting nitrification is therefore increased by the presence of the calcium salt, potassium carbonate and calcium sulfate being formed.
=416. Production of Nitrous and Nitric Acids.=—In the following pages the study of the methods of isolating the nitrous and nitric ferments will be considered as one process, the final isolation of the two classes of bodies being the result of their synchronous cultivation in appropriate media. The special process of the production of ammonia by oxidation is not so well-known, and will therefore be described in brief.
It is now generally conceded that the action of the nitrous organism is precedent to that of the nitric, but the two processes go on so nearly together as to prevent the accumulation of any large quantities of the lower salt in the soil.
Whether or not the formation of ammonia precedes that of nitrous acid is still a subject for experimental demonstration. Chemically, both nitrous acid and ammonia may be produced by the reduction of nitric acid. In nature, the reverse of this process may be the customary method.
=417. Production of Ammonia in the Soil by the Action of Microbes.=—It is highly probable that organic nitrogen in the soil in passing into the form of nitric acid exists at some period of the process in the form of ammonia.
Marchal has isolated and studied some of these ammonia-making bacteria.[276] Bacillus mycoides is the most active of these organisms. It occurs constantly in surface soils and is present in the air and in natural waters. In decomposing albumen it produces a strongly alkaline solution due to ammonium carbonate. Organic carbon, during this process, is converted chiefly into carbon dioxid, but small quantities of formic, propionic, and butyric acids are also produced. Any organic sulfur which is present is converted into acid. No hydrogen or nitrogen is eliminated in a free state. While slight alkalinity is favorable to the development of this bacterium, yet it may be propagated in a feeble sulfuric acid solution when the acid is less than one per cent.
The greatest activity of this organism is manifested at 30°. Below 5° and above 42° no ammonia is produced. The bacillus will not develop in an atmosphere of hydrogen or carbon dioxid, except in solutions of organic matter and nitrate. In addition to its action on egg albumen it decomposes other proteid bodies as well as leucin, tyrosin, creatin, and asparagin. It, however, does not oxidize urea, nor does it develop in solutions of ammonium salts and nitrates, except as mentioned above. When soluble carbohydrates are present, acids are formed. It is concluded from these experiments that the final oxidation of organic nitrogenous matter is preceded by its conversion into ammonium carbonate.
=418. Summary of Statements.=—All nitrogenous matters which would be naturally present in the soil may become subject to nitrification when the proper conditions are supplied. Munro has also succeeded in nitrifying ethylamin, thiocyanates, and gelatin, urea, asparagin, and the albuminoids of milk and rapeseed.
The products of nitrification are ammonia, nitrous or nitric acid, carbon dioxid, and water. The ammonia and nitrous acid may not appear in soils as the final products of nitrification, as the nitric organism attacks the latter at once and converts it into nitric acid. Nitrous acid and ammonia may also be produced in soils as one of the retrograde steps in denitrification.
To summarize the conditions necessary for nitrification it may be said that first, the proper material must be supplied; viz., an organic or inorganic nitrogenous compound capable of oxidation. In the second place, the medium must be faintly alkaline, the temperature must not be too high, the nitrifying organisms must have abundant food, and the process must take place in the dark.
=419. Order of Oxidation.=—It is quite definitely determined that activity of the ammoniacal and nitrous organisms is the first step in the process, since the nitric organism appears to have no power whatever to oxidize proteid compounds; while, on the other hand, the nitrous organism can not, in any case, complete the conversion of nitrous into nitric acid.
The conditions which permit certain organisms to oxidize free nitrogen have not been definitely determined. The presence of such bodies in the tubercles attached to the rootlets of certain leguminous plants has been established. Lately, Winogradsky has isolated from the soil a nitrifying organism which is capable of converting free nitrogen into forms suited to nourish plant growth. This organism is cultivated in dextrose with careful exclusion of all nitrogen, save that which exists in the air carefully freed of every trace of ammonia or oxidized nitrogen.
Under the influence of the growth of this organism the sugar undergoes a butyric fermentation, and nitrogen in an oxidized form is assimilated in an amount apparently equal to about one five-hundredth of the sugar consumed.
This result leads Warington[277] to remark that it is a fact of extraordinary interest, both to the physiologist and chemist, that a vegetable organism should be able to acquire from the air all the nitrogen it needs.
=420. The Nitrification of Ammonia.=—The same organism which converts organic nitrogen into nitrous acid acts also on ammonia and its compounds with a similar result. In fact, the formation of ammonia may be regarded as one of the stages on the road from albuminoid to nitric nitrogen.
Data have been collected by Schloesing on the nitrification of ammonia taking place in arable soil, tending to show that this phenomenon is accomplished without appreciable loss of nitrogen in the gaseous state.[278] This, however, does not hold good when the quantity of ammonium carbonate introduced into the earth is largely increased. In two experiments, conducted by Schloesing, with a larger quantity of ammonium carbonate, the loss of nitrogen was very notable. In certain conditions the production of nitrous acid may take place, and it is interesting to know whether the appearance of nitrites has any influence on the disengagement of free nitrogen. In order to determine this question a solution of calcium nitrite was prepared by decomposing silver nitrite with calcium chlorid. From the results of the experiments made it was seen that the nitrites were only the results of a retarded and partially incomplete nitrification. They are, moreover, thus an obstacle to the normal work of the nitrifying organisms. It is also established that when they are present a disengagement of gaseous nitrogen takes place, whether the nitrites are formed during the progress of the experiment, or whether they were originally present. However, it is not best to say that the nitrites themselves have been the cause of the disengagement of the nitrogen. It may happen that the disengagement of the nitrogen and the presence of nitrites are simply simultaneous and due to one and the same cause. The destruction of nitrates in the midst of reducing agents furnishes, according to the nature of these bodies and the circumstances, nitrous acid, nitrogen dioxid, nitrogen protoxid, free nitrogen, and even ammonia.
This destruction of nitrates and the appearance of oxids of nitrogen and of free nitrogen are more likely to be due to the presence of a separate denitrifying ferment as pointed out by Springer than to have arisen in the manner mentioned above by Schloesing. In the present state of our knowledge, moreover, we can hardly regard the presence of nitrites as an obstacle to complete nitrification. On the other hand, it seems to be well established that the production of nitrites or ammonia is a necessary step between organic nitrogen and nitric acid.
=421. Occurrence of Nitrifying Organisms.=—According to the observations of Schloesing and Müntz the nitrifying organisms are widely distributed.[279] Arable soil containing considerable humus seems to be the medium in which they grow most freely and in which they accomplish their most important functions. Sewage waters are also rich in nitrifying ferments, and, in fact, all waters containing organic matter. They are also found in running waters but not in great numbers. They affect chiefly the surface of bodies, and especially are found on the bottom of culture-flasks.
These authors have not found the nitrifying organisms in normal air. They could not seed sterilized flasks by admitting air freely. The absence of these ferments from the air is explained by reason of their sensitiveness to desiccation.
The method used by Schloesing and Müntz for the separation of the organism consisted in the preparation of original and subcultures in sterilized solutions containing nitrifiable matters. The proof of isolation was assumed when a given subculture contained only one kind of organism as seen with the microscope. The appearance of this organism, as described by the authors, was that of the later isolations by Warington and Winogradsky, but the method used could hardly now be regarded as decisive.
=422. Determination of Nitrifying Power of Soils.=—In studying the distribution of the nitrifying organisms in a soil the general method of procedure is based on the production of nitrification in a convenient solution by the organisms present in a given sample of soil. If the solution seeded with the given portion of soil remain unaffected, it will show that there were no nitrifying organisms present in the seed used. On the other hand, the vigor of the nitrifying process when once it is started, may be taken as an evidence of the number and activity of the organisms in the soil, a sample of which was used for seed.
=423. Composition of the Culture Medium.=—The solution recommended by Warington for the culture and isolation of the nitrifying ferments has the following composition:
Ammonium chlorid 80 milligrams. Sodium potassium tartrate 80 „ Potassium phosphate 40 „ Magnesium sulfate 20 „ calcium carbonate about 200 „ Pure bacteria-free water to make one liter.
=424. Apparatus and Manipulation.=—The experiments are conducted in short, wide-mouthed bottles. The initial volume of the solution in each bottle is 100 cubic centimeters, and the bottle should be of such size as to give a depth of liquid of from three to five centimeters.
The neck of the bottle is closed with a plug of cotton and this is protected from dust by tying over it a cap of filter paper. Arranged in this way, filtered air has free access to the solution. The bottle with the solution thus protected is placed in a water-oven and kept near the temperature of boiling water for six to eight hours to destroy any organisms present. When cool, the solution is ready for use.
The calcium carbonate used should be prepared by precipitation and added in a moist state. The calcium carbonate solution should be added after the sterilization of the liquid, the precipitated carbonate being boiled just before it is added.
Preparation of Seed.—The seed employed to start the nitrification should be a small quantity of fresh soil, usually about one-tenth of a gram. If a previously nitrified solution be used for seed it should be thoroughly shaken and about one cubic centimeter of the solution removed for seeding the new bottle.
In introducing the nitrifying liquor into the bottle the plug should be lifted slightly and a small pipette inserted by means of which the liquor is added. The operation should be carried on in a room perfectly free from dust and to which no one but the operator has access. The greatest care should be exercised to prevent any particles of matter entering the solution except that which is purposely added. In withdrawing the liquor from the nitrifying solution cotton wool should be pressed around the top of the pipette so that the entering air may be filtered before admission to the interior of the bottle. The pipette which is used should be kept in boiling water until it is required for use. After use it should be washed and replaced in boiling water until again required.
After seeding, the bottles should be placed in a dark cupboard and exposed to the ordinary temperature of the laboratory. If a higher or stated temperature be desired, the bottle should be placed in a metal box the temperature of which can be regulated to any degree.
Test of the Commencement of Nitrification.—The beginning of the nitrification can be determined in a solution by testing it with diphenylamin. One cubic centimeter of the solution withdrawn as above indicated, is placed in a small beaker, a drop of solution of diphenylamin sulfate in sulfuric acid added, and then two cubic centimeters of concentrated sulfuric acid and the contents of the beaker well shaken. The development of a violet-blue color shows the presence of nitric or nitrous acid. This test will detect one part of nitric nitrogen in twenty million of water.
Determining the Progress of Nitrification.—The progress of nitrification is determined by repeated examinations for ammonia by nesslerizing, and for nitrous acid with metaphenylenediamin. Each experiment is made with five cubic centimeters of the solution withdrawn as above indicated and placed in test-tubes, always of the same size. The reaction with the nessler solution is then made by adding it in the usual way. The colorations are recorded as, trace, small, moderate, considerable, large, and abundant.
If the change produced by the organism consisted in the formation of nitrites only, the ammonia in the original solution would fall from large to trace, while the nitrous acid would increase from trace to large. If the nitrification consisted in the production of nitrates only, the ammonia would diminish without any corresponding production of nitrous acid. In mother solutions which contain ammonium carbonate instead of sulfate, it should not be forgotten that the ammonia might gradually disappear owing to the volatilization of the carbonate without any corresponding production of free nitrites or nitrates. The complete disappearance of the ammonia in the above experiments shows the completion of the process.
=425. To Determine the Distribution of the Nitrifying Organism in the Soil.=—The principle on which the determination of the distribution of the nitrifying organism in the soil depends, rests upon seeding the growth solutions with samples of soil taken at different depths and carefully protected from the time of sampling until the time of seeding from any admixture of accidental organisms.
The method of Warington is the simplest and best to follow.[280] The samples of soil are taken by digging a pit of convenient depth usually from eight to ten feet. A fresh surface is then cut on one of the sides of the pit at the spot selected for sampling. This surface is scraped with a freshly ignited platinum spatula. The spatula should then be washed, re-ignited, and cooled, and a small portion of the soil, at the depth required, detached with the spatula and transferred at once into one of the growth bottles already described.
The growth solution best suited for the purpose contains four cubic centimeters of urine per liter. Each bottle should also contain some freshly precipitated calcium carbonate. In sterilizing urine solutions the calcium carbonate should be added before the heating instead of afterwards. The quantity of soil taken for each seeding should be about one-tenth of a gram.
Inasmuch as the cotton stopper has to be lifted to introduce the soil, opportunity is given for the entrance of any organisms floating in the air. Experience, however, has shown that air free from soil dust very seldom contains nitrifying organisms. The seeded bottles are placed in a dark cupboard of moderate temperature as already described.
=426. Sterilized Urine Solution.=—The sterilized urine solution used for the determination of the distribution of the nitrifying organisms in the soil, is made by taking four cubic centimeters of healthy urine, diluting to one liter, adding some freshly precipitated calcium carbonate, stoppering with cotton wool and heating for several hours at the boiling temperature of water.
As a result of Warington’s experiments it was shown that the nitrifying organism in the soil did not exist, at least in portions of one-tenth of a gram, to a greater depth than eighteen inches. In only one case was nitrification produced from a sample of soil taken at a greater depth and this may have been due to the accidental introduction of organisms from other sources. It may be assumed that any long delay in the commencement of nitrification under favorable conditions, implies the presence of a very limited quantity of organisms in the solution. Thus a comparative study of the period of incubation and the progress of nitrification in solutions seeded with soils taken at different depths or at different places, becomes a fair index of the number and vitality of the nitrifying organisms contained therein.
=427. Depth to Which Micro-Organisms are Found.=—Koch states that at the depth of about one meter, the soil is nearly free from every kind of bacteria.[281] These observations have been corroborated by Pumpelly and Smyth who find that no infection of a bacterial nature is produced in a sterilized solution from samples of clay taken at the depth of nine feet below the surface.[282]
It is evident from the nature of the experiments above described that the nitrifying processes go on almost exclusively in those portions of the soil which are subject to cultivation, while in the subsoil and below the processes of nitrification are either retarded or arrested. Any stores of nitrogenous matter, therefore, in an insoluble state, resting in the subsoil, are preserved from oxidation and consequent waste until such time as they may be removed to near the surface.
=428. Isolation of the Nitrous and Nitric Organisms in the Soil.=—The action of the organisms which produce nitrification either in form of nitrites or nitrates, having been thoroughly established, and the method of testing the soil therefor given, it remains to describe a method by means of which these organisms in the soil may be isolated and obtained in a state of purity. The difficulties attending this process are extremely great on account of the similarity of the two organisms. All earlier attempts to make pure cultures of the two separate organisms were attended with but little success.
According to Winogradsky the method of culture on gelatin so long practiced is not to be relied upon.[283] It is very difficult to eliminate by this process the organisms which grow rapidly in gelatin and which mature their colonies in two or three days, but where they require eight or ten days to produce a colony the method is successful. In fact, by the gelatin process as it was at first practiced, a good deal was owing to chance, but sometimes by a happy accident a pure nitro-bacterium might be isolated.
Formerly it was considered that a liquid could be regarded as sterile if it gave no growth upon gelatin. It has, however, now been demonstrated that a liquid may contain large numbers of nitro-bacteria and still produce no growth upon gelatin. However, for the organisms which accompany the nitro-bacteria in soils, it is regarded as certain that if no growth on gelatin is produced by them they are absent. Therefore in the case of a solution which has been seeded with a soil, if it can be brought to such a state as to produce no growth on gelatin, it may be safely assumed that it contains no bacterial organisms save those which are capable of producing nitrites or nitrates. Therefore if such a solution produce nitrification and at the same time no growth upon gelatin, it may be considered as a proof of the isolation of the nitro-organisms from all others.
This method was also worked out independently by Mr. and Mrs. Frankland.[284]
Winogradsky says further he confesses that he has advanced these views only provisionally and without being convinced of their infallibility. Strictly speaking, the proof of seeding gelatin is not sufficient alone because the absence of growth can not be regarded as the exclusive privilege of the nitro-bacteria. Such might be the case sometimes for an accidental mixture of microbes, introduced with any given sample of soil into the cultures, but the criterion is not absolute. Microbes, for example, of a sulfurous or ferruginous nature may be cited, for which the gelatin layer is not only unfavorable but even fatal. It may thus happen that there may be eliminated from the solution all that will grow upon gelatin without freeing it from some special kinds of cultures, refractory like the nitro-bacteria, but which might reappear if they should be resown in some favorable nutritive solution. On account of this fault in the process, Winogradsky has been impressed with the necessity of bringing out a better method.
In using the gelatin media it is necessary to find the one that is suited to nourish these organisms, which would evidently be the way promising the greatest success. This having been found, and those organisms which produce colonies being easily recognizable, a great step towards the solution of the problem will have been made and the more so as the medium would be at the same time absolutely unfavorable to other forms of microbes. On account of the slow degree of development of the nitro-organisms, all others would probably have opportunity to grow and strengthen to their exclusion, unless these interfering organisms could be completely removed.
=429. The Culture Solution.=—The culture-solution, first proposed by Winogradsky, had the following composition:
To ten grams of gelatin or one part of agar-agar in 100 cubic centimeters of water add potassium phosphate, one-tenth of a gram; magnesium sulfate, five-hundredths of a gram; calcium chlorid, trace; and sodium carbonate, half a gram. The solution being sterilized in the usual way by heating, there are added to it a few cubic centimeters of a sterilized solution containing two-tenths per cent of ammonium sulfate. Such a solution has been proved to be very favorable to nitro-organisms. Nevertheless the experiments with such solutions gave no definite results and they were abandoned.
The non-success of this method led Winogradsky to adopt a nitrifying solution which absolutely excluded all organic substances. Instead of using an animal or vegetable gelatinous substance he used one of a mineral nature, first proposed by Graham and Kühne.[285] Two of these gelatinous mineral substances were considered; viz., the aluminum hydroxid and the hydrate of silica. The latter was chosen.
=430. Preparation of the Mineral Gelatinous Solution.=—The soluble glass which is found in commerce is generally of a thick, sirupy consistence. It is first diluted with three times its volume of water. One hundred cubic centimeters of this liquid are poured with constant stirring into fifty cubic centimeters of dilute hydrochloric acid and the mixture placed in a dialyzer. It is useless to employ a standard solution of silica. All that is necessary is to submit to dialysis a liquid with an excess of acid and sufficiently dilute not to be exposed to the danger of being spontaneously gelatinized in the dialyzer. The dialyzer is left for one day in running water and two days in distilled water, often renewed. The solution is then ready for use. This is the case when it is no longer rendered turbid on the addition of silver nitrate, showing that the hydrochloric acid has been entirely extracted. The solution is then to be sterilized by boiling, and preserved in a glass flask closed with a plug of cotton.
More recent instructions by Winogradsky for preparing the gelatinous silica recommend dialyzing the soluble glass after treatment with hydrochloric acid in a parchment tube.[286] The proportions of silicate and acid are 100 cubic centimeters of the silicate solution (1.06 specific gravity) and 100 cubic centimeters of hydrochloric acid (1.1 specific gravity). With a dialyzing tube placed two days in running water and one day in distilled water frequently changed it will be found that the acid is completely removed. One hundred cubic centimeters of the residual liquor giving no reaction for hydrochloric acid are concentrated to twenty cubic centimeters. When cold there is added one cubic centimeter each of a solution of ammonium sulfate and of sodium carbonate, together with corresponding quantities of the other nutrient salts commonly employed. The ammonium sulfate should never exceed two to two and a half, and the sodium carbonate four parts per thousand. To the flask containing the above substances is added one drop of the seed-liquor, which may be a soil water or a drop from some previous culture. The flask is shaken and the mixture poured into a low circular glass dish which is covered by one slightly larger in diameter (Petri double dish). To the liquid in the dish is added a drop of a cold saturated solution of common salt, and it is then stirred with a platinum spatula. The addition of the salt greatly favors the setting of the jelly. The jelly may set in from two to three hours, but a longer time secures better results in the end.
In employing these preparations as seed, after the organisms have grown, it is absolutely necessary to use the isolated cellules and not the aggregated masses (zoöglœæ). The latter are rarely free of foreign germs which adhere to their gelatinous envelope. Since the zoöglœæ can not be broken up by any artificial means it is necessary to await their spontaneous disintegration in order to separate the mobile monads. The opalescence of the culture-liquid is a sure index of this separation.
The particles of mineral gelatin to be used as seed for nitrifying are best taken as follows:
A glass tube is drawn out immediately preceding the operation, until the end is as fine as a hair. The surface of the mineral gelatin is magnified by means of a dissecting microscope magnifying 80 to 100, to the proper degree and the preparation table is so arranged as to give a perfect support to the right hand which should hold the filament of glass. The smallest colony is then pricked with the needle and the end of the glass is broken and dropped into the flask which is to be seeded. The seed is thus selected in as small a particle as may be desired, only a few cells, but it can always be ascertained with certainty that some of the particles have been obtained by this operation.
The method of cultivation on mineral jelly is considered by Winogradsky an important resource in the study of the nitrifying organisms. It removes the chief difficulties heretofore existing in discovering and characterizing these organisms among the innumerable micro-organisms of the soil. The long series of cultures necessary to separate the organisms are rendered nugatory. By directly introducing a little of the earth into the silicic jelly the active organisms in nitrification can be at once discovered. It is preferable, however, as indicated below, to previously produce a nitrification in an aqueous solution by a trace of earth and to take from it the seed for impregnating the solid medium. In order to show at once a proof of its nitrifying character, it is only necessary to take a small bit of the mineral jelly, the size of a grain of rye, and to throw it into a little sulfuric acid which has been treated with diphenylamin. There is at once formed a blue spot equal in intensity to a saturated solution of anilin blue.
In regard to the growths which nitro-organisms make in a medium of the kind described, they are far from being so marked as are those produced by ordinary micro-organisms.
A nitro-bacterium is not capable of the energy of growth which is recognized for the greater number of microbes. The colonies contained in the gelatin always remain small. The largest among them are just visible to the naked eye like white points. Along the striae, on the contrary, there is formed quite a thick white crust. To the naked eye, in general, there is nothing very characteristic in the formation of colonies in a medium of this nature. But this impression changes altogether when the placques are examined with a low magnifying power. The colonies, especially those of the interior surface, reveal then an aspect so curious as to be well remembered when once seen.
This mineral gelatin, as has already been noticed, is very unfavorable to the growth of microbes other than nitro-bacteria and becomes altered only under the action of the air. If the placques be carefully preserved from desiccation the culture of these organisms can be continued for several weeks. Although they do not seem to increase, the colonies, as well as the jelly, are still in a good condition at the end of that time. Nevertheless the expectation that this medium would prevent the formation of any foreign organism has not been realized. Some of the organisms which accompany the nitro-bacteria in soil, also grow upon the silicic jelly; but they do not form colonies, properly so-called, and their growth is extremely slow. They generally make their appearance before the nitro-bacteria and spread exclusively upon the surface in form of white spots, so transparent that without careful examination they would not be discovered. Having reached a certain size the spots do not change during entire weeks. This circumstance renders the operations of isolation somewhat delicate, but does not prevent them.
=431. Preparation and Treatment of the Solution to be Nitrified.=—The organisms having been grown on the siliceous gelatin in the manner described they are tested for their nitrifying power as follows:
The mineral solution which is to be nitrified with the above preparation is composed of ammonium sulfate, four-tenths gram; magnesium sulfate, half a gram; potassium phosphate, one-tenth gram; calcium chlorid, trace; sodium carbonate, six-tenths to nine-tenths gram; and distilled water, 100 cubic centimeters. The sulfates with the calcium chlorid on the one hand, and the phosphate and carbonate on the other, are dissolved separately and the two solutions sterilized separately and mixed after cooling. The seeding is then done as described above.
=432. Isolation of the Nitrous and Nitric Organisms.=—Instead of proceeding immediately to the isolation of special organisms in the soil, the preliminary period of purification is prolonged by Winogradsky by allowing the free growth to take place of all the organisms which can be maintained in the ordinary medium.[287]
The composition of the culture solution employed is as follows: Distilled water, 1,000 parts; potassium phosphate, one part; magnesium sulfate, half a part; calcium chlorid, trace. Each flask receives besides this some magnesium carbonate, freshly washed with boiling water and added in slight excess.
The flasks thus charged are sterilized, and after sterilization there are added two cubic centimeters of a solution of two per cent of ammonium sulfate, which, when added to fifteen or twenty cubic centimeters of liquid give from two to two and a half parts per thousand.
They are then seeded with soil. The reasons for this preliminary treatment are as follows: First, all the observations upon the enfeeblement of the oxidizing power of these organisms have been made upon cultures seeded simply by the fresh soil, and in cultures derived therefrom. In the second place, the existence of the two forms, one nitrous and the other nitric, prevents at once the isolation of a single organism.
Samples of soil from Europe, Africa, Asia, Australia, and America, were used for seed for the experiments. First, the cultures were made by seeding with a small quantity of each of these samples of soil, and each one of these cultures served as a point of departure for a series of subcultures. The temperature of the cultures should be kept constantly at 30°.
The method of following the nitrification adopted by Winogradsky is essentially that of Warington, the percentage of ammonia remaining at any time being determined by nesslerizing. To detect the presence of nitric acid the nitrous acid is decomposed by boiling with ammonium chlorid in excess, or with urea, and then diphenylamin is used as a reagent. By treatment with ammonium chlorid and boiling, the ammonium nitrite is resolved into free nitrogen and water as indicated by the equation NH₄NO₂ = N₂ + 2H₂O. Or the total oxidized nitrogen may be estimated by the Schloesing method or by any of the standard methods hereafter given. The nitrous acid is then determined by potassium permanganate and the nitric acid by difference.
A great difference is to be noted between freshly taken earth and that which has been kept for a long while, especially when sealed. With fresh earth taken near the surface a mere trace is sufficient to produce nitrification. With samples of earth which have been kept for a long while and thoroughly dried, several grams must be added in order to secure perfect nitrification. The period of incubation with the samples of earth ranges from three to twenty days. The beginning of the phenomenon is revealed by the appearance of nitrous acid, of which the quantity is increased very rapidly, but in the end it disappears and is transformed into nitric acid.
=433. Statement of the Results.=—The method of stating the results of examination of soils for nitrifying organisms is illustrated by the following example:
Soil from Zurich. The culture was seeded on the 11th of October, one gram of soil being taken. On the 20th of October the nitrous acid had reached its maximum of intensity and there was no ammonia left. On the 29th of October the nitrous acid remained almost stationary and there was hardly any nitric acid present. On the 1st of November the reaction for nitrous acid began to decrease. On the 5th of November the reaction for nitric acid was very intense. On the 11th of November the nitrous acid had all disappeared except a mere trace.
The above order of phenomena was observed with all the samples of soil tried, from which it is concluded with certainty that nitrifying organisms transplanted directly from their natural medium in the soil into a liquid easily nitrifiable produce at once nitrous acid in abundance. The phenomenon of nitrification is divided into two periods therefore, of which the first is devoted to the production of nitrites, and the second consists in the oxidation of the nitrites, and this does not commence until the total disappearance of the ammonia. Occasionally the formation and oxidation of the nitrites practically go on together, but never equally, the oxidation of the nitrites being always sensibly behind their formation.
=434. Method for Subcultures.=—From the mother cultures described above, Winogradsky makes subcultures as follows:
The solution to be nitrified is prepared as in the mother cultures. The seeding is accomplished by adding a small quantity of the liquor of the mother culture after shaking. Subcultures can be made in this way to the seventh generation.
In respect of the oxidation of the nitrites the results may be entered as negative if they have not disappeared at the end of two months.
To determine whether the process of oxidizing the nitrites is in progress or not the total nitrous acid is estimated, and the process repeated at the end of eight or ten days. Should there be no diminution of the nitrous acid within this time it may be considered that the further oxidizing action is not taking place.
=435. Use of a Solid Medium.=—It may be justly claimed that the action of nitrifying organisms in a liquid is not to be compared with their action in a solid medium, such as a soil which is their natural habitat. It might be, therefore, that the inability of the nitrous organism to produce nitrates is due to the nature of the medium in which it is cultivated. Winogradsky in order to determine this question cultivated the organism in a solid medium of two kinds, first a silicate gelatin impregnated with an ammonium salt and second in sterilized earth. The silicate jelly is prepared as follows:
Mix a jelly of silica containing some ammonium sulfate with sterilized soil. The seeding is done with one of the subcultures which no longer has the power of producing nitrates.
In the case of the jelly the seeding is accomplished as follows:
A minute drop of a culture liquid is taken with a capillary glass tube and applied in striae to different parts of the solid jelly; or a minute drop of the culture liquid may be mixed with the jelly before solidification. The Petri dishes in which these cultures are made can be preserved in a moist atmosphere and thus the desiccation be easily prevented for a long time. From time to time small pieces of the jelly as large as a pea can be taken and tested for the progress of nitrification.
Results.—The nitrous reaction, both in the prepared jelly and in sterilized soil, will appear in a few days. At the end of from seven to twelve days it will have attained its maximum intensity and will then remain stationary indefinitely. Sterilized soil has no power to generate the nitric from the nitrous ferment. The two organisms are, therefore, of different species.
After a few generations the power of producing nitrates seems to be lost although the nitrous ferment may still be active. This suppression of the power to oxidize the nitrites is not due to any pernicious influence of the culture-medium but to the condition of the successive solutions at the time of taking the seeding samples.
=436. Microscopic Examination.=—A small particle of the deposit in the culture-liquid is spread on a glass slide and dried. There is then added a drop of very dilute perfectly transparent malachite green solution. Malachite green is Bittermandelölgrün, or tetramethyldiamidotriphenylcarbinol. Use the zinc chlorid double salt or oxalate. In about half a minute it is washed and colored by a very dilute solution of gentian violet which is left to act for some time. The cells then appear distinctly colored on a colorless background.
In examining in this way nitrous cultures under a moderate enlargement there are seen particles of material covered with scattered groups and massive zoöglœæ composed of cells which are, doubtless, identical.
By their round or roundish forms, by their relative size and especially by their numbers and uniformity they are at once distinguished from the other vegetations which are generally of a purely bacillus shape.
With the exception of some shreds of mycelium coming from some oidium in the soil the microscope reveals nothing but the organisms described. The microscopic appearance[288] of the nitrous ferment is shown in Fig. 69.
The general conclusions of Winogradsky are:
1. Each soil possesses but one organism capable of oxidizing ammonia.
2. Soils from one locality have always the same kind of nitrifying ferment.
3. Soils from different and distant countries contain nitrifying organisms which differ from one another in some respects so much so that it may be necessary to distinguish a few species or even genera in these bodies.
=437. Isolation of the Nitric Ferment in Soils.=—The principle of the separation of this ferment as described by Winogradsky rests upon the fact that in culture solutions of a mineral nature free from ammonia the nitrous ferment will not grow, whereas if nitrite or nitrous acid be present the nitric ferment will grow.[289] In a few generations, therefore, the nitrous ferment will be entirely eliminated.
Solution employed:
Distilled water 1,000 grams. Potassium phosphate 1 gram. Magnesium sulfate 0.5 „ Calcium chlorid trace. Potassium nitrite 0.22 gram.
To culture-flasks containing 100 cubic centimeters of the above mixture after sterilization about one-tenth gram of fresh soil is added. In favorable conditions the nitrous acid will disappear in about fifteen days.
Subcultures are made by seeding fresh portions of the sterilized solution with one or two cubic centimeters of the mother culture. The operation is continued until the nitrous ferment is eliminated.
The organisms in the deposit in the culture-flasks are then subjected to microscopic examination in the manner already described for the nitrous ferment; or proceed as follows:
=438. Culture on Solid Media.=—Take a liquid which has been employed in the culture of a nitrous ferment and evaporate to one third of its bulk. Gelatinize the residue by adding double its volume of the silicic acid solution prepared as already directed.
The jelly is placed in the glass vessels usually employed. The seeding may be done with a few drops of a culture-liquid containing the nitric ferment as obtained above. The first reaction will appear in from eight to ten days. In about forty-five days the nitrous acid in the jelly will have entirely disappeared. Two classes of colonies are noticed under the microscope. The first to appear are small colonies which never extend beneath the surface of the jelly. In cultures seeded with these colonies there is no oxidation of nitrous acid. The second class of colonies extends into the interior of the jelly. They are much larger than the first, of a yellowish-gray color and not spherical but rather lenticular in shape. Cultures seeded with these colonies will lose their nitrous acid in about ten days or two weeks.
The growth of these organisms in a liquid scarcely merit the name of cultures. The naked eye can usually distinguish no form of vegetation. The liquid remains clear, the surface is free from any film, no flocks are deposited. Colored and examined in the microscope the organisms found are so puny as to make doubtful their oxidizing power. There is an apparent contradiction between the powerful chemical action that these organisms can produce and their apparent deficiency in physical properties.
These organisms are best found by cultivating them in a very limpid solution. The bottoms of the culture boxes will be found covered with an extremely tenuous gelatinous deposit communicating to the glass a feeble grayish-blue tint. The culture bottle is inclined and the bottom scratched with a recently drawn-out capillary tube. The colonies rise in the tube together with a little of the liquid. The colonies are dried, mounted, and colored as already described and when examined with the microscope are found to be composed exclusively of masses of an organism of extreme minuteness.
The organism remains attached so firmly to the bottom of the culture bottle that it can be washed several times with pure water without danger of detachment and thus rendered more pure.
In old cultures which are sustained by new additions of nitrite an extremely transparent pellicle on the bottom of the flask can be distinguished. By shaking the liquid some fragments may be detached and made to float through the fluid. With a little care and patience these flocks can be captured, mounted, and colored. Since they show the nitric organism in its natural state their preparations are of the greatest interest.
The best preparations are made by coloring with malachite green and gentian violet and then coloring again hot with magenta. Afterwards the preparation is washed with warm water at 50°–60° which takes almost the whole of the color from the gelatinous matter. The cells are then clearly presented colored a reddish violet on a rose background. These organisms[290] are shown in figure 70.
The figure shows the cells united by a gelatinous membrane and grouped in small dense masses composed often of a single layer of organisms. The cells are generally elongated, rarely regularly spherical or oval. Their mean length does not exceed half a micromillimeter and their thickness is from two to three times less.
The difference in form of the nitrous and nitric ferments is very marked and leaves no doubt of the existence of these two forms which are as distinct as could be desired in microbic discrimination.
=439. Dilution Method of Warington.=—The method pursued by Warington in preparing pure cultures of the nitrifying ferment is based on the well-known principle of dilution which may be expressed as follows:[291] In a liquid containing bacterial ferments dilution may be practiced until a drop of the liquid may be taken which will contain no more than a single organism of any one kind. If now proper solutions be seeded with single drops of this solution, some of them may give colonies of pure cultures of any given organism. The solution to be nitrified employed by Warington had the following composition:
Water 1000 parts. Ammonium carbonate 0.25 „ Ammonium chlorid 0.50 „ Potassium phosphate 0.04 „ Magnesium sulphate 0.02 „ Calcium sulphate 0.02 „
The ammonium chlorid is added to prevent the precipitation of magnesium and calcium phosphates. The solution is kept in wide-mouthed, stoppered bottles to prevent the loss of ammonium carbonate, the bottles being only half full. About 100 cubic centimeters are taken for each experiment. These bottles are sterilized and seeded with fresh soil in the ordinary way. They are then covered with paper caps and placed in a dark cupboard at a constant temperature of 22°.
Special Media.—A quantity of arable soil is exhausted of nitrates by washing with cold water under pressure. The soil is then boiled with water and filtered. The clear amber-colored solution obtained may be used instead of water in the above formula.
Solid Media.—(1) Ordinary ten per cent gelatin made with beef broth and peptone. (P)
(2) A ten per cent urine solution solidified with six per cent of gelatin. (U)
(3) A solution of one gram of asparagin, one-half gram sodium acetate, one-half gram potassium phosphate, two-tenths gram magnesium sulfate, two-tenths gram calcium sulfate, and one liter of water solidified with six per cent gelatin. (As)
Other solid media may also be employed for the purpose of favoring, as much as possible, the growth of the nitrifying organisms.
The first culture in the ammonium carbonate solution given above, is always made by seeding with a little unmanured arable soil. Subcultures are seeded from this mother culture by seeding new solutions with a few drops of the original. In all cases tried by Warington the subcultures produced only nitrous fermentation while the original cultures produced the nitric fermentation.
=440. Microscopic Examination.=—The microscopic examination of the organisms formed is conducted as follows:
The cover glasses for microscopic objects are placed at the bottom of the culture-flask, the cover glasses being previously sterilized. At the end of the nitrification the liquid is removed with a pipette and the flask containing the cover glasses dried at 35°. The cover glasses are then removed and stained. The microscopic appearance of the organisms obtained by the previous cultures showed masses of corpuscles usually of oval shape and having a length generally exceeding one micromillimeter. An immersion objective giving a magnification of 800 diameters is suitable for this work.
Other forms of organisms are also met, the whole series being characterized as follows:
(1) The corpuscles already mentioned. Larger ones are frequently rough in outline resembling masses of siliceous sea-sand. The smaller oval corpuscles are regular in form.
(2) Some very small circular organisms often appearing as mere points and staining much more plainly than the preceding.
(3) A few slender bacilli, staining faintly.
All the cultures obtained by the above method give abundant growth on gelatin.
=441. Trials with the Dilution Method.=—One part of the third subculture in the ammonium carbonate solution described above, is mixed with 500 parts of thoroughly boiled water and one drop from a sterilized capillary tube is added to each of five bottles containing the sterilized ammonium carbonate solution. In Warington’s experiments one of the five bottles was found to have nitrified after forty-one days. After ninety-one days two more were nitrified. Two bottles did not nitrify at all. All three solutions which nitrified gave growths on gelatin. The growths took place more speedily on gelatin U and As than on P.
The organisms obtained on gelatin were seeded in appropriate liquid media but no nitrification was obtained.
A subculture from solution No. 2 of the first dilution mentioned above, was diluted to one one-thousandth, one ten-thousandth, one one-hundred-thousandth, and one one-millionth. Each of these dilutions was used for seeding with five sterilized solutions of ammonium carbonate, using the method of seeding above described. At the end of 190 days not one of these solutions had nitrified.
Warington supposed that the cause of failure in the method just mentioned might be due to the alkalinity of the ammonium carbonate. While this solution could be seeded in the ordinary way with fresh earth it might be that the faint alkalinity which it presented might prevent it altogether from action when the nitrifying agent was reduced to a few organisms.
He therefore changed the solution to one of the following composition:
Water 1,000 parts. Ammonium chlorid 0.02 part. Potassium phosphate 0.06 „ Magnesium sulfate 0.03 „ Calcium sulfate 0.03 „
The solution was divided in twenty stoppered bottles which were half filled. The bottles were divided into four series, A, B, C, D, each one consisting of five bottles, and these were respectively seeded with one drop from dilutions to one one-thousandth, one ten-thousandth, one one-hundred-thousandth, and one one-millionth of a second subculture of No. 3 in the first dilution series.
After 115 days, nitrification had occurred in ten of the bottles. The other ten did not nitrify at all. Each of the nitrifying solutions was spread on gelatin, P and U being employed. Growth took place far more easily on gelatin U than on gelatin P. Of the ten nitrified solutions there were three which gave no growth on gelatin U, either when spread on the surface or introduced into the substance of the jelly. There were therefore secured nitrifying solutions which did not contain organisms capable of growing on gelatin. The supposition is therefore fair that they were pure nitrifying organisms. These fresh, pure organisms had the faculty of converting ammonia into nitrous acid only and not into nitric acid.
With the organisms thus prepared a number of solutions of potassium nitrite containing phosphates and other mineral ingredients were seeded. In no case was any loss of nitrite found, which is proof that the solution contained no organisms capable of oxidizing nitrous acid. The organisms prepared as above, have the power of nitrifying organic substances containing nitrogenous bodies.
The organism isolated as described and examined under the microscope is seen to contain two forms. The first one is nearly spherical in shape, the corpuscles varying in size from mere points to a diameter of one micromillimeter. The form is very striking and easily stained. The second form is oval-shaped and attains a length distinctly exceeding one micromillimeter. Sometimes it is a regular oval and sometimes it is egg-shaped. This form is stained less easily than the preceding or spherical form.
=442. Method of Staining.=—The method of staining employed is as follows:
A drop of the culture-liquid is placed on a glass slide and mixed with the filtered stain by means of a wire. A cover glass is placed on the drop and allowed to stand for half an hour. It is then pressed down on the slide and the liquid which exudes wiped off and hollis glue run around the cover glass. In this way the organism is stained in its own culture-fluid and can be seen in its true form without any possibility of the destruction of its shape by drying. The plate is bright and clear though colored.
If the preparation is to be mounted in balsam a drop of the culture is dried in the center of a cover glass. It is then placed for some minutes in dilute acetic acid to remove matter which would cause turbidity. The cover glass with its contents is then washed, dried, and stained for some hours in methyl violet.
=443. Classification of Nitrifying Organisms.=—The names proposed by Winogradsky for the various organisms are the following:
For the general group of microbes transforming ammonia into nitric acid, Nitro-bacteria.
For the nitrous ferments of the Old World
Genus, Nitrosomonas:
Species, Nitrosomonas europaea.
Nitrosomonas javanensis.
For the nitrous microbes of the New World:
Genus, Nitrosococcus.
Species, not determined.
For the nitric ferment:
Genus, Nitrobacter.
=444. Nitrification in Sterilized Soil.=—The process of nitrification in sterilized soil, when seeded with pure cultures, is determined as follows:
Preparation of Sample.—The fresh sample of arable soil is freed from pebbles and vegetable débris and reduced to as fine a state of subdivision as is possible in the fresh state. It is placed in quantities of about 800 grams in large crystallizing dishes.
One dish is set aside for use in the natural state, and the other, hermetically closed, is placed in a sterilizing apparatus and subjected to the action of steam for two and a half hours. This treatment is repeated three times on as many successive days.
Seeding of Sample.—Each of the two dishes is moistened with fifty cubic centimeters of pure water containing 500 milligrams of ammonium sulfate. The sterilized portion is then seeded with a preparation of the pure nitrous ferment, produced as before described. The seed is prepared by filtering a few cubic centimeters of the nitrous culture liquid through asbestos. The asbestos is well washed and then thrown into a flask containing a few cubic centimeters of sterilized water and well shaken. The water carrying the filaments of asbestos is poured drop by drop on the surface of the soil in as many places as possible. The two dishes of soil are kept at an even temperature of 20° in a dark place. Winogradsky found that, treated in this way, the unsterilized soil produced only nitrates, while the sterilized portions produced only nitrites.[292]
=445. Variation of the Determinations.=—To vary the conditions of the experiment Winogradsky uses twelve flasks of the erlenmeyer shape, four having bottoms twelve centimeters in diameter, and eight of them five centimeters in diameter. In each of the four large flasks are placed 100 grams of fresh soil, and in each of the eight small flasks twenty-five grams. The eight small flasks are designated a, b, c, d, and a′, b′, c′, d′, and the four large flasks A, B, C, D.
The flasks a, b, c, d, and a′, b′, c′, d′, are placed in a stove at 30° for several days before use, while A, B, C, and D, are kept at 22°–23° for the same length of time. The soil in the small flasks is, therefore, somewhat drier than that in the large ones.
The flasks are treated as follows:
a, a′, A, contain the soil as prepared above for control.
b, b′, B, are sterilized at 135° and seeded with a drop of the pure nitrous culture.
c, c′, C, sterilized as above and seeded with a little of the unsterilized earth.
d, d′, D, sterilized as above and seeded with pure nitrous and pure nitric cultures.
After sterilization there was added to the small flasks two cubic centimeters of a twenty per cent sterilized ammonium sulfate solution, and to the large ones six cubic centimeters. At the end of a month or six weeks the contents of the flasks are thrown on a filter and washed with cold water until a drop of the filtrate gives no blue color with diphenylamin. The respective quantities of nitrite and nitrate are then determined in the filtrates by the usual processes, which will be fully described further along.
=446. Sterilization.=—One of the chief requisites for success in the bacteriological investigation of soils is found in the thoroughness of the sterilizing processes. The value of cultures depends chiefly on the care with which the introduction of foreign germs is prevented. In the following description a mere outline of the method of sterilization is presented, while those who wish to study more carefully the details of the process are referred to the standard works on bacteriology.
=447. Sterilization of the Hands.=—It is important that the hands of the operator handling apparatus and materials for bacteriological work should be sterilized. The sterilization may be accomplished in the following way:
The nails should be cut short and thoroughly cleaned with soap and brush. The hands are thoroughly washed in hot water with soap. After washing in hot water the hands should be washed in alcohol and ether. They are then dipped in the sterilizing solution.
This liquid may consist of a three per cent solution of carbolic acid, which is the one most commonly employed. A solution of corrosive sublimate, however, is perhaps the best disinfectant. It should contain from one to two parts of the crystallized salt to 1,000 parts of water. It has lately been advised to use the sublimate in an acid solution. Acetic acid or citric acid may be employed, but hydrochloric acid is recommended as the best, in a preparation of one-half part per 1,000. For stronger solutions of sublimate containing more than a half per cent, equal quantities of common salt should be added. The solution should be made with sterilized water.
After dipping the hands in the sterilizing solution they should be dried with a napkin taken directly from a sterilizing oven, where it has been kept for some time at the temperature of boiling water. Where only ordinary work in bacteriology is contemplated this sterilization of the hands is not necessary. It is practiced chiefly in antiseptic surgery.
=448. Sterilizing Apparatus.=—With platinum instruments the most effective and easiest way for sterilizing is to hold them in the flame of a bunsen until they are red hot. Steel and copper instruments can not be treated in this way without injury. They are best sterilized by submitting them to dry heat in a drying oven at a temperature of 150°–160° for two hours. Glass and porcelain apparatus can be sterilized best in the same way.
All apparatus and materials employed should be used in a space as free as possible from dust, so that any germs which might be carried in the dust can be excluded from the apparatus in transferring it from one place to another.
=449. Methods of Applying Heat.=—Sterilization by means of heat may take place in several ways.
First. Submitting the Materials to Dry Heat Without Pressure.—The temperature in sterilization of this kind may vary from the temperature of boiling water at sea-level to 160° obtained by an oil-bath or by an air-oven.
Second. Sterilization in a Liquid Under Pressure.—This form of sterilization may be effected by sealing the liquid in a strong vessel and submitting it to the required temperature. If the temperature required be greater than that of boiling water the vessel can be immersed in a solution of some mineral salt which will raise the boiling-point.
Third. Sterilization in Steam Under Pressure.—This method of sterilization consists in placing the body in a proper receptacle in vessels to which the steam can have access and then admitting steam from a boiler at any required pressure. In the case of small apparatus, such as the autoclave, the steam can be generated in the apparatus itself. The variety of apparatus used in the above method of sterilization is very great, but all the forms of apparatus employed depend upon the principles indicated.
=450. The Sterilizing Oven.=—The apparatus for sterilization by means of hot, dry air usually consists of a double-walled vessel made of sheet-iron, usually with a copper bottom. The apparatus is shown in Fig. 71.
The temperature is controlled by means of a thermometer, T, and the gas-regulator, R. This is one of the ordinary gas-regulators by means of which the amount of gas supplied to the lamp is increased if the temperature should fall, and diminished if it should rise above the required degree. The best form of the sterilizing ovens is provided with a means for circulating the hot air so that the temperature may be made uniform throughout the mass. This can be accomplished by introducing a mechanical stirrer, or by the movement of the air itself.
Between the walls of the vessel may be placed water, provided the temperature of sterilization be that of boiling water. If it should require a higher temperature than boiling water, a solution of salt can be added until the required temperature is reached, or the space between the two walls may be left vacant and hot air made to circulate around the oven.
The exterior of the oven, except at the bottom where the lamp strikes the copper surface, should be protected by thick layers of asbestos or other non-conducting material. To avoid danger of flying filaments, this covering should be coated with some smooth paint which will leave an even surface not easily abraded.
=451. Sterilization with Steam at High Pressure.=—The apparatus used for this is commonly called an autoclave and is shown in Fig. 72.
The top is movable and held in place by the clamp, a, which is fixed by the screw worked by the lever, b. The vessel itself is double-jacketed and the pressure is obtained from water in the vessel heated by means of the lamp, c. The actual steam pressure is indicated by the index d. The safety-valve, e, allows any excess of steam to escape above the amount required for the maintenance of the pressure. This, however, is best regulated by the lamp. The outer jacket permits the heat from the lamp to circulate around the inner pressure vessel, and the holes near the top, oo, are for the escape of the heated gases. Enough water is placed in the bottom of the inner pressure vessel to supply all the aqueous vapor necessary to produce the required pressure and still leave some water in excess.
The materials to be sterilized are held on the shelves of the stand and the vessels may be of various kinds according to the nature of the material to be sterilized. The vessels containing the material being covered, the steam does not come in actual contact with it. At the end of the operation the safety-valve must not be opened to allow the escape of the steam, otherwise the remaining water would be rapidly converted into vapor and would be projected over the materials on the shelves. The lamp should be extinguished and the apparatus allowed to cool. The autoclave is not only useful for sterilizing purposes but can be made of general use in the laboratory where heat under pressure, as in the estimation of starch, etc., is required.
These two forms of apparatus are sufficient to illustrate the general principles of sterilization by hot air and steam. There are, however, many variations of these forms designed for special use in certain kinds of work. For full descriptions of these, reference is made to catalogues of bacteriological apparatus.
=452. Arnold’s Sterilizing Apparatus.=—A very simple and cheap steam sterilizer has been devised by Arnold.
Water is poured into the pan or reservoir, B, Fig. 73, whence it passes through three small apertures into the shallow copper vessel, A. It is there converted into steam by being heated with any convenient lamp, and rises through the funnel in the center to the sterilizing chamber. Here it accumulates under moderate pressure at a temperature of 100°. The excess of steam escapes about the cover, becomes imprisoned under the hood, E, and serves to form a steam-jacket between the wall of the sterilizing chamber and the hood. As the steam is forced down from above and meets the air it condenses and drips back into the reservoir. Such an apparatus as this is better suited to commercial purposes, as the sterilizing of milk, than for scientific uses.
=453. Thermostats for Culture Apparatus.=—It is important in the culture of micro-organisms that the temperature should be kept constant during the entire time of growth. Inasmuch as some operations continue for as much as three months it is necessary to have special forms of apparatus by means of which a given temperature, during the time specified, can be maintained. This is secured by means of an oven with an automatic temperature regulator, practically built on the principle of the hot air sterilizing oven already described.
The essential principles of construction are, however, that the regulator for the temperature should be delicate and that the non-conducting medium surrounding the apparatus should be as perfect as possible, so that the variations in temperature from changes in the exterior temperature, are reduced to a minimum. This delicacy is secured by introducing a drop of chloroform-ether into a confined space over the mercury of the regulating apparatus. The doors of the chamber are double, the interior one being of glass so that the exterior door can be opened for inspection of the progress of the bacterial growth without materially interfering with the interior temperature. A convenient form is shown in Fig. 74.
The apparatus figured, is oval in shape, although circular or other forms are equally as effective. The arrangement of the lamp, a, thermometers, t t t, and gas-regulator, g, and the double doors, d d, is shown in the engraving and does not require further description.
The usual temperatures for cultures range from 22° to 35°, and the apparatus once set at any temperature will remain fixed with extremely minute variations for an indefinite time. The apparatus possesses a heat zone which, by the arrangement of the regulator, is kept absolutely constant. The space between the walls of the apparatus being filled with water, the temperature is maintained even in every part. The apparatus, as constructed, is independent not only of the surrounding temperature within ordinary variations, but also of the pressure of the barometer. Three thermometers are employed to determine the temperature of the heating zone, the water space and the inner space. The arrangement of the gas-regulator is of an especial kind, as mentioned above, by means of which the consumption of gas is reduced to a minimum. This apparatus can be regulated to suit the character of the work.
=454. Microscopic Apparatus Required.=—Any good microscope, capable of accurate observation, of high power, may be used for the bacteriological observations necessary to soil analysis.
Preference should be given to the patterns adapted to receive any additional accessories which may be subsequently required for advanced work. The stage, in addition to being fitted with a sliding bar, should have a large circular or horseshoe opening to facilitate focusing operations. A mechanical stage is a desirable acquisition if really well made, but a plain stage is preferable for many purposes. A rackwork, centering sub-stage is essential for advanced work, and in the absence of the more complete form, there should at least be a fitting beneath the stage to take the diaphragm and condenser. An iris diaphragm will be found more useful than any other kind in practice, since the size of the opening can be increased very gradually at will.
One of the best lamps is known as the paraffin lamp and is fitted with a half-inch wick. This will give even more light than is actually required, and a steady flame, perfectly under control, may be obtained. For the minute details to be observed in high-grade microscopic work, such as is required in the bacteriological examination of soils, reference must be had to the standard works on bacteriology and microscopy.
=455. General Conclusions.=—The nitrogenous food of plants is provided in several ways; viz., (1) By the nitrogen brought to soil in rain and snow. This nitrogen is chiefly in the form of ammonia and nitric acid. The nitrogen gas in solution in rain water has no significance as a plant food. (2) By the action of certain anaerobic organisms herding in the rootlets of leguminous plants, free nitrogen may be oxidized and put into form for assimilation. (3) By the action of certain organisms on nitrogenous compounds pre-existing in the soil, ammonia, nitrous acid, and finally, nitric acid, are produced. It is believed that the plant organism, unaided by the activity of a micro-organism, is unable to assimilate nitrogen unless it be fully oxidized to nitric acid. (4) There exist micro-organisms capable of acting directly on free nitrogen independent of other plant growth, but the significance of this possible source of plant food is, at the present time, unknown. (5) The micro-organisms of importance to agriculture may be isolated and developed to the exclusion of other organisms of a similar character. This isolation is best accomplished in culture-media consisting essentially of a mineral gelatin to which is added only pure carbohydrates and the necessary mineral nourishment. (6) The nitrifying ferments consist probably of several species, of different geographic distribution. Different types of soils probably have nitrifying organisms of different properties. This is illustrated by the fact that nitrification is accomplished in dry alkaline soils under conditions in which the ordinary nitrifying organisms would fail to develop. (7) The study of typical soils in respect of the kind, activity, and vigor of their nitrifying organisms has become as important a factor in soil analysis as the usual determination of physical and chemical composition.
DETERMINATION OF NITRIC AND NITROUS ACIDS IN SOILS.
=456. Classification of Methods.=—The minute quantities in which highly oxidized nitrogen exists in soils render the operations of its quantitative estimation extremely delicate. On the other hand, the easy solubility of these forms of combination and the absence of absorptive powers therefor, in the soil, render the separation of them from the soil a matter of great ease. It is possible, therefore, to secure all the nitrates and nitrites present in a large quantity of earth in a solution which can be concentrated under proper precautions to a volume convenient for manipulation. The method of this extraction is the same for all the processes of determination. The methods of analysis suited to soil extracts, as a rule, may also be used in the determination of the same compounds in rain, drainage, and sewage waters, and for the qualitative and quantitative control of the progress of nitrification. The various processes employed may be classified as follows:
1. The conversion of the nitrogen into the gaseous state and the determination of its volume directly. This is accomplished by combustion with copper oxid and metallic copper.
2. The conversion of the nitrogen into nitric oxid and the volumetric determination thereof. The decomposition of a nitrate with ferrous chlorid in the presence of free hydrochloric acid is an instance of this type of methods.
3. The oxidation of colored organic solutions and the consequent disappearance of the characteristic color, or its change into a different tint. The indigo and indigotin processes are examples of this method.
4. The production of color, in a colorless or practically colorless solution, by the treatment thereof with the nitrate in presence of an acid which decomposes it with the liberation of oxidizing compounds. The depth of color produced is compared with that formed by a known quantity of a pure nitrate solution until the two colorations are alike. The methods depending on the use of carbazol or acid phenol sulfate illustrate this class of reactions.
5. The conversion of the nitrogen into ammonia by moist combustion with sulfuric acid in the presence of certain organic compounds, e. g., salicylic acid, and the collection of the ammonia in standard acid, the excess of which, is determined by titration.
6. The reduction of nitrates to ammonia by nascent hydrogen and the recovery of the ammonia produced by distillation and collection in standard acid.
7. The reduction of nitrates by electrolytic action and the collection of the ammonia as above.
8. The decomposition of nitrates with the quantitative evolution of a different element, and the direct or indirect measurement of the evolved substance. The quantitative evolution of chlorin on treating a nitrate with hydrochloric acid, the collection of the chlorin in potassium iodid, and the determination of the iodin set free, form a process belonging here.
=457. Relative Merit of Methods.=—The processes mentioned in the classifications embraced under numbers (1) and (5) of the preceding schedule are sufficiently described in the paragraphs devoted thereto, under soil and fertilizers. In practice at the present time it is rare to determine the nitrogen in nitrates by the copper oxid method. The more rapid and equally exact processes of colorimetric comparison or reduction by nascent hydrogen are in all respects to be preferred.
The indigo methods among the colorimetric processes are not so much in use now as those which depend on the development of a color. Lawes and Gilbert considered them far inferior to the Schloesing method. The developed color methods are especially delicate and are to be preferred in all cases where the detection of the merest traces of nitrates is desired. Where nitrates are present in considerable quantities the reduction method with nascent hydrogen is to be preferred over all others. In all these cases the judgment of the analyst must be exercised. The particular method to be employed in any given case can not be determined save by the intelligent discrimination of the operator.
=458. The Extraction of Nitric Acid from the Soil.=—The easy solubility of nitric acid and of nitrates in water is taken advantage of in the separation of these bodies from the soil. A convenient quantity, usually about 1,000 grams of the fine soil, is taken for the extraction. Instead of freeing the soil entirely from water, it is better to determine the amount of water in the air-dried or prepared sample, and base the calculation on 1,000 grams of the water-free soil.
All samples of soil, when taken for the purpose of examining for nitrates, should be rapidly dried to prevent the process of nitrification from continuing after the sample is taken. For this purpose the soil should be placed in a thin layer in a warm place, 50°–60°, until air-dried. It still contains in this case a little moisture but not enough to permit nitrification to go on.
One thousand grams of soil prepared as above are treated with 2,000 cubic centimeters of distilled water, free of nitric acid, and allowed to stand for forty-eight hours with frequent shaking. One thousand cubic centimeters of the extract are then filtered, corresponding to 500 grams of the dry soil. A small quantity of pure sodium carbonate should be added to the filtrate which is then evaporated on a water-bath to a volume of about 100 cubic centimeters. Should a precipitate be formed during evaporation it should be separated by filtration, the filter washed thoroughly, and the filtrate again evaporated to a volume of 100 cubic centimeters.
In taking a soil for the determination of nitrates, it is well to extend the sampling to a considerable depth. If the sample be taken only to the depth of nine inches, it should be in dry weather when the nitrates are near the surface.
The temperature at which a soil is dried has also an influence on the quality of nitric nitrogen remaining after desiccation.
If a wet soil be dried at 100°, the nitrates present will suffer partial decomposition. This is probably due to deoxidation by organic matter present. On the other hand, ordinary air-drying affords opportunity for continued nitrification, thus increasing the residuum of oxidized nitrogen. The above method is essentially that followed by Warington at Rothamstead.
The method of drying practiced at Rothamstead, in order to secure results as nearly accurate as possible is the following:[293]
The soil is broken up directly after it is taken from the field, and spread on trays in layers one inch deep. The trays are then placed in a room at 55°. The drying is completed in twenty-four hours. After drying, stones and roots are removed, and the soil is finely powdered and placed in bottles.
For extracting the nitrates, a funnel is prepared by cutting off the bottom from a bottle four and a half inches in diameter. A nicely fitting disk of copper gauze is placed in the bottom of this funnel, and this is covered with two filter papers, the upper one having a slightly greater diameter than the lower. The paper is first moistened, and then from 200 to 500 grams of the dry powdered soil introduced. The funnel is connected with the receiving flask of a filter pump, and pure water poured on the soil until it is thoroughly saturated. The water is then added in small quantities. When the filtrate amounts to 100 cubic centimeters the process may be discontinued, since all the nitrates in the soil will be found in this part of the filtrate.
The extract obtained above is evaporated to convenient bulk for the determination of nitric nitrogen.
THE NITRIC OXID PROCESS.
=459. Method of Schloesing.=—The processes for estimating nitrogen by combustion with copper oxid and by moist combustion with sulfuric acid have both been used for the determination of the quantity of nitrogen existing in a highly oxidized state. These processes will be fully discussed under the head of fertilizers. In the case of soil extracts, drainage waters, etc., it will be sufficient to discuss, for the present, only those processes adapted especially to a quick and accurate estimation of oxidized nitrogen.
The principle of the method of Schloesing depends on the decomposition of nitrates in the presence of a ferrous salt and a strong mineral acid.[294] The nitrogen in the process appears as nitric oxid, the volume of which may be directly measured, or it may be converted into nitric acid and titrated by an alkali.
The typical reactions which take place are represented in the following equation:
6FeCl₂ + 2KNO₃ + 8HCl = 3Fe₂Cl₆ + 2KCl + 4H₂O + 2NO.
=460. Schloesing’s Modified Method.=—The Schloesing method as now practiced by the French chemists is conducted in the apparatus shown in Fig. 75.[295] The carbon dioxid is generated by the action of the hydrochloric acid in F on the fragments of marble in A. After passing the wash-bottle the gas enters the small tubulated retort, C, which contains the nitrate in solution. For ordinary soils 100 grams are placed in an extraction flask, plugged with cotton, and a layer of the same material is placed over the soil for the purpose of securing an even distribution of the extracting liquid. This liquid is distilled water containing in each liter one gram of calcium chlorid. The purpose of using the calcium chlorid is to prevent the soil from becoming compacted which would render the extraction of the nitrate difficult. The extracting liquid is allowed to fall drop by drop from a mariotte bottle until the filtrate amounts to 500 cubic centimeters. This volume is concentrated on a sand-bath until it is reduced to ten or fifteen cubic centimeters when it is transferred to a flat-bottomed dish and the evaporation finished over steam, care being taken not to allow the temperature to exceed 100°.
Another and more rapid method for dissolving the nitrate, may also be practiced. In a flask holding about one liter, place 220 grams of the soil and 660 cubic centimeters of distilled water and shake vigorously, or enough water to make 660 cubic centimeters together with the moisture remaining in the air-dried sample taken. All the nitrates pass into solution. Throw the contents of the flask into a filter and take 600 cubic centimeters of the filtrate which will contain all the nitrates in 200 grams of the sample taken. This filtrate is evaporated as described above.
In the flat dish containing the dried nitrates, pour three or four cubic centimeters of ferrous chlorid solution and stir with a small glass rod until complete solution of the nitrate takes place. By means of a small funnel the solution is poured into C, and the capsule and funnel are well rinsed with two cubic centimeters of hydrochloric acid. The washing is repeated three times as above described, and once with one cubic centimeter of water, which is added cautiously so as to form a layer over the surface of the heavier liquid. The tubulated flask is then connected with the carbon dioxid apparatus, previously freed from air, and the gas allowed to flow evenly until the whole of the apparatus is completely air-free. The other details of the method are essentially the same as those adopted by the Commission of French Agricultural Chemists which will be given below.
=461. The French Agricultural Method.=—The Schloesing method as practiced by the French agricultural chemists is very slightly different from the procedure just described.[296] The process with soils is carried on as follows:
Five hundred grams of the soil are taken and introduced into a flask of about two liters capacity and shaken thoroughly with a liter of distilled water. The whole of the nitrates of the soil is thus passed into solution. The solution is filtered and 400 cubic centimeters of the filtrate are taken, which correspond to 200 grams of the soil. This liquid is evaporated in a flask, adding a fragment of paraffin to prevent foaming, until its volume is reduced to fifteen or twenty cubic centimeters. It is afterwards transferred through a filter into a capsule with a flat bottom in which the evaporation is finished on a steam-bath, taking care that the temperature does not exceed 100°. An important precaution is, not to allow the contact of the water with the soil to be too prolonged, to avoid the reduction of the nitrates which could take place under the influence of the denitrifying organisms which are developed with so great a rapidity in moist earth. The apparatus in which the transformation of the nitrates into nitric oxid takes place is essentially that already described (Fig. 75). The carbon dioxid generator is connected by means of a rubber tube and a small wash-bottle to the small retort in which the reaction takes place, and from which the exit tube leads to a mercury trough. The gas which is disengaged is received under a jar drawn out to a fine point in its upper part, which carries about fifteen cubic centimeters of potash solution containing two parts of water to one of potash.
The operation is conducted as follows:
Into the small capsule which contains the dried matter, three or four cubic centimeters of ferrous chlorid are poured. By means of a stirring rod the residue sticking to the sides of the capsule is detached with care and all the matter is thus collected in the bottom. By means of a small funnel the contents of the capsule are introduced into the retort. About two cubic centimeters of hydrochloric acid are used for washing out the materials and this acid is also introduced into the retort. The washing with hydrochloric acid is repeated three or four times, and finally the apparatus is washed with one cubic centimeter of water, which is also poured in by the small funnel with great care, so that this water may form a layer over the surface of the liquid. The apparatus is now connected and filled completely with carbon dioxid. Since it is necessary that this gas should be completely free of air, the flask, which generates it, is first filled with the acidulated water from the acid flask, and the air is thus almost totally displaced by the liquid. The evolution of carbon dioxid gas which follows, completely frees the apparatus from air. When this is accomplished the retort is connected with the rest of the apparatus and the gas allowed to pass for about two minutes until the air is completely driven out of all the connections. The current is arrested for a moment by pinching the rubber tube which conducts the carbon dioxid into the retort, and the vessel which is to receive the gas is then placed over the delivery-tube, this vessel being filled with mercury and a strong solution of potash. The communication between the retort and the carbon dioxid flask is broken and the flask is heated slightly by means of a small lamp. The first bubbles of gas evolved should be entirely absorbed by the potash. This will be an indication of the complete absence of the air. When the liquid is in a state of ebullition the nitrogen dioxid is set free. The boiling is regulated in such a way that the evolution is regular and the liquid of the retort may not, by a too violent boiling, pass into the receiver. The boiling is continued until the larger part of the liquid is distilled and only three or four cubic centimeters remain in the retort. At this time a few bubbles of carbon dioxid are allowed to flow through in order to cause to pass into the receiver the last traces of nitric oxid. The gas received is left for some minutes in contact with the potash.
Afterward in a small flask, G, the neck of which is drawn out to a fine point, and carrying a bulb-tube, H, and a piece of rubber tubing, there are boiled twenty-five or thirty cubic centimeters of water for five or six minutes in order to drive all the air out of the flask, and while the boiling is continued the rubber tubing is fastened to the drawn-out part of the jar containing the nitric oxid. Within the rubber tubing the drawn-out point is broken and the vapor of water is forced into the jar and drives before it the solution of potash which has filled the capillary part of the drawn-out tube. As soon as the point is broken, the boiling of the flask is stopped and by its cooling the nitric oxid passes into it. It is necessary to press the rubber tubing with the fingers in order that the passage of the gas into the flask be not too rapid. As the solution of potash rises in the bell-jar which contains the nitric oxid near to the point where the rubber tubing covers its drawn-out portion, the fingers are removed and a clamp put in their place. There still remains a little nitric oxid in the flask and to drive this out it is necessary to introduce five or six cubic centimeters of pure hydrogen, which are allowed to pass over into the receiving flask, by releasing the clamp in the same way as the nitric oxid. The hydrogen being introduced in successive portions, finally carries all the nitric oxid into the flask without allowing any of the potash to enter.
The flask containing the nitric oxid is now connected with a reservoir of oxygen. The oxygen is allowed to enter, bubble by bubble, by means of cooling the flask by immersion in water. The transformation of nitric oxid into nitric acid is not entirely complete for twenty-four hours. It is necessary, therefore, to wait that long after the introduction of the oxygen before determining the amount of nitric acid produced.
The contents of the flask are placed in a titration-jar, the flask being washed two or three times and a few drops of tincture of litmus being added. The nitric acid is then determined by a standard solution of calcium hydroxid or some other standard alkali. From the titration the content of nitric acid is calculated.
The French Committee further suggests that this method may be modified in the way of making it more rapid by collecting the nitric acid in a graduated tube filled with mercury and containing some potash. The volume of the gas is determined and the pressure of the barometer and the temperature observed, and the usual calculations made to reduce the volume to zero and to a pressure of 760 millimeters of mercury. Each cubic centimeter of nitric oxid thus measured corresponds to 2.417 milligrams of nitric acid. The presence of organic matter does not interfere with the determination of nitric acid by either of the methods given above.
=462. Modification of Warington.=—The method of procedure and description of apparatus used, as employed by Warington, are as follows:
The vessel in which the reaction takes place is a small tubulated receiver, A (Fig. 76), about four centimeters in diameter, mounted and connected as shown in the illustration. The delivery-tube dips into a jar of mercury in a trough containing the same liquid. The long supply funnel-tube a is of small bore, holding in all only one-half cubic centimeter. The connecting tube F, carrying a clamp, is also of small diameter and serves to connect the apparatus with a supply of carbon dioxid.
In practice, the supply-tube a is first filled with strong hydrochloric acid and carbon dioxid passed through the apparatus until the air is all expelled. This is indicated when a portion of the gas collected over the mercury, is entirely absorbed by caustic alkali.
At this point the current of carbon dioxid is stopped by the clamp C, and a bath of calcium chlorid, B, heated to 140° is brought under the bulb A, until the latter is half immersed therein. The temperature of the bath is maintained by a lamp. By allowing a few drops of hydrochloric acid to enter the receiver, the carbon dioxid is almost wholly expelled. The end of the delivery-tube is then connected with the tube, T, filled with mercury, and the apparatus is ready for use.
The nitrate, in which the nitric acid is to be determined, in a dry state, is dissolved in two cubic centimeters of the ferrous chlorid solution (one gram of iron in ten cubic centimeters), one cubic centimeter of strong hydrochloric acid is added, and the whole is then introduced into the receiver through the supply-tube, being followed by successive rinsings with hydrochloric acid, each rinsing not exceeding one-half cubic centimeter. The contents of the receiver are, in a few moments, boiled to dryness; a little carbon dioxid is admitted before dryness is reached, and again afterwards to drive over all remains of nitric oxid. In the recovered gas the carbon dioxid is first absorbed by caustic potash, and afterwards the nitric oxid by ferrous chlorid. All measurements of the gas are made in Frankland’s modification of Regnault’s apparatus. The carbon dioxid should be as free as possible from oxygen. The carbon dioxid generator is formed of two vessels, the lower one consisting of a bottle with a tubule in the side near the bottom; this bottle is supported in an inverted position and contains the marble from which the gas is generated. The upper vessel consists of a similar bottle standing upright and containing the hydrochloric acid required to act on the marble. The two vessels are connected by a glass tube passing from the side tubule of the upper vessel to the inverted mouth of the lower vessel. The acid from the upper vessel thus enters below the marble. Carbon dioxid is generated and removed at pleasure by opening a stop-cock attached to the side tubule of the lower vessel thus allowing hydrochloric acid to descend and come in contact with the marble. A good Kipp’s generator of any approved form may also be used instead of the simple apparatus, above described.
The fragments of marble used are previously boiled in water in a strong flask. After boiling has proceeded for some time, a rubber stopper is fixed in the neck of the flask and the flame removed. Boiling will then continue for some time in a partial vacuum.
The hydrochloric acid is also well boiled and has dissolved in it a moderate quantity of cuprous chlorid. As soon as the acid has been placed in the upper reservoir, it is covered by a layer of oil. The apparatus being thus charged is at once set in active work by opening the stop-cock of the marble reservoir; the acid descends, enters the marble reservoir, and the carbon dioxid produced drives out the air. As the acid reservoir is kept on a higher level than the marble reservoir, the latter is always under internal pressure, and leakage of air from without, into the apparatus, cannot occur.
The presence of the cuprous chlorid in the hydrochloric acid not only insures the removal of dissolved oxygen, but affords an indication to the eye of the maintenance of this condition. While the acid remains of an olive tint, oxygen is absent; but should the color change to a blue-green, more cuprous chlorid must be added. All the reagents employed should be previously boiled.
In order to secure absolute freedom from air, the following modifications on the above process have been adopted by Warington: The apparatus having been mounted as described, the funnel-tube attached to the bulb retort is filled with water, and the apparatus connected with the carbon dioxid generator. Carbon dioxid is then passed through the apparatus until a moderate stream of bubbles rises in the mercury trough. The stop-cock is left in this position, and the admission of gas is controlled by the pinch-cock. The bath of calcium chlorid is so adjusted as to cause the bulb retort to be almost entirely submerged, and the temperature of the bath is kept at 130° to 140°. Small quantities of water are next admitted into the bulb and expelled as steam in the current of carbon dioxid, the supply of this gas being shut off before the evaporation is entirely completed, so as to leave as little carbon dioxid as possible in the apparatus. Previous to very delicate experiments it is advisable to introduce through the funnel-tube a small quantity of potassium nitrate, ferrous chlorid, and hydrochloric acid, rinsing the tube with the latter reagent. Any trace of oxygen remaining in the apparatus is then consumed by the nitric oxid formed; and after boiling to dryness and driving out the nitric acid with carbon dioxid, the apparatus is in a perfect condition for a quantitative experiment.
=463. Preparation of the Materials to be Analyzed.=—According to Warington, soil extracts may be used without other preparation than concentration.
Vegetable juices which coagulate when heated, require to be boiled and filtered or else evaporated to a thin sirup, treated with alcohol, and filtered. A clear solution being thus obtained, it is concentrated over a water-bath to a minimum volume in a beaker of small size. As soon as cool, it is mixed with one cubic centimeter of a cold saturated solution of ferrous chlorid and one cubic centimeter of hydrochloric acid, both reagents having been boiled and cooled immediately before use.
In mixing with the reagents, care must be taken that bubbles of air are not entangled, which is apt to occur with viscid extracts.
The quantity of ferrous chlorid mentioned is amply sufficient for most soil extracts, but it is well to use two cubic centimeters in the first experiment, the presence of a considerable excess of ferrous chlorid in the retort being thus insured. With bulky vegetable extracts more ferrous chlorid should be employed. To the sirup from twenty grams of mangel-wurzel sap, five cubic centimeters of ferrous chlorid and two cubic centimeters of hydrochloric acid are usually added.
=464. Measurement of the Gas.=—The measurement of the gas was for some time conducted by the use of concentrated potash for absorbing the carbon dioxid, and ferrous chlorid for absorbing the nitric oxid. The use of the ferrous chlorid, however, was found to introduce a source of error. The treatment of the gas with oxygen and pyrogallol over potash has therefore been substituted by Warington for its absorption by ferrous chlorid.
The chief source of error attending the oxygen process lies in the small quantity of carbon monoxid produced during the absorption with pyrogallol; this error becomes negligible if the oxygen be only used in small excess. The amount of oxygen employed can be regulated by the use of Bischof’s gas delivery-tube. This may be made of a test-tube having a small perforation half an inch from the mouth. The tube is partly filled with oxygen over mercury, and its mouth is then closed by a finely perforated stopper made from a piece of wide tube and fitted tightly into the test-tube by means of a covering of rubber. When this tube is inclined, the side perforation being downwards, the oxygen is discharged in small bubbles from the perforated stopper, while mercury enters through the opening. Using this tube, the supply of oxygen is perfectly under control and can be stopped as soon as a fresh bubble ceases to produce a red tinge on entering. Warington concludes his description by stating that in the reaction proposed by Schloesing the analyst has a means of determining a very small quantity of nitric acid with considerable accuracy, even in the presence of organic matter; but to accomplish this, the various simplifications consisting in the omission of the stream of carbon dioxid, and the collection of the gas over caustic soda must be abandoned, and special precautions must be taken to exclude all traces of oxygen from the apparatus.
=465. Spiegel’s Modification.=—Spiegel noticed inaccuracies in the results of the ferrous chlorid method of estimating nitric acid when carbon dioxid is used, which sometimes amounted to three per cent of the nitric acid present in the sample. The following suggestions are made by him for the improvement of the process:[297]
As regards the use of carbon dioxid in the operation, the first difficulty consists in obtaining it entirely free from air. By the use of small pieces of marble, which, before being placed in the Kipp apparatus, are kept for a long while in boiling water, a product is obtained which, after thirty minutes of moderate evolution, leaves only a trace of unabsorbed gas in contact with potash-lye. The apparatus used is illustrated in Fig. 77.
A is a round flask of about 150 cubic centimeters capacity, furnished with a well-fitting rubber stopper provided with two holes, one for the entrance of the funnel-tube B and the other for the delivery-tube C. The tube B ends about two centimeters above the bottom of A and carries a bulb-shaped funnel at its top capable of holding about fifty cubic centimeters. The gas-tube D is ground into the bulb of B as shown in the figure.
After the flask had been filled with the solution to be examined, carbon dioxid is conducted through D and the flask is heated to boiling until the gas which escapes through C no longer contains any air. The measuring tube is brought over the end of the delivery-tube C, in the usual manner, but not shown in the figure. In the funnel of B are placed twenty cubic centimeters of previously prepared and boiled ferrous chlorid solution and this liquid is allowed to flow partly into A by lifting slightly the gas-tube, D. About forty cubic centimeters of concentrated, boiled hydrochloric acid are afterwards added to it in the same way. As soon as the liquid in the flask A is again boiling, the stream of carbon dioxid is shut off and allowed to flow again only towards the end of the operation, when the contents of the flask are reduced almost to dryness. As will be seen from the above directions no unboiled liquids of any kind are to be used as reagents in the apparatus described. If the flask A were made much smaller the efficiency of this apparatus would be increased. It appears to have few, if any, advantages over Warington’s process.
=466. Schulze-Tiemann Method.=—The modification of Schulze-Tiemann in the ferrous salt method consists chiefly in the omission of the use of carbon dioxid, and in the simplified form of apparatus, which permits rapid work and gives, also, according to some authorities, very exact and reliable results.[298] The extract, representing 500 grams of the fine soil, is reduced by evaporation to 100 cubic centimeters and placed in a glass flask, A (Fig. 78), of 500 cubic centimeters capacity. The flask is closed with a rubber stopper, carrying two bent glass tubes which pass through it. The tube a b c is drawn out into a point at a and reaches about two centimeters below the surface of the rubber stopper. The tube e f g passes just to the lower surface of the rubber stopper. The two tubes mentioned are connected, by means of rubber tubes and pinch-cocks, with the tubes d and h. The pinch-cocks at c and g must be capable of closing the tubes air-tight. The end of the tube g h passes into a crystallizing dish, B, and is bent upward to a point passing two to three centimeters into the measuring tube C. The point within the tube is covered with a piece of rubber tubing. The measuring tube C is divided into tenths of a cubic centimeter, and together with the crystallizing dish B, is filled with a ten per cent solution of boiled soda-lye, which is obtained by dissolving 12.9 parts of sodium hydroxid in 100 parts of water.
The liquid which is to be examined for nitric acid, the pinch-cocks being opened and the tube g h not dipping into the crystallizing dish, is boiled for one hour in order to drive the air out of the flask A. The end of the tube e f g h is then brought into the crystallizing dish containing the sodium hydroxid solution so that the steam escaping from the flask A, escapes partly through the tube b c d, and partly through the tube f g h, not allowing, however, the bubbles to enter the measuring tube C. To determine whether the air is all expelled, the pinch-cock at g is closed and the soda-lye will thereupon rise to g in case no air interferes. It is best to close the tube at g first with the thumb and finger and then the rise of the soda-lye to that point can be determined by the impulse felt. The tube is then firmly closed by means of the pinch-cock g. The rest of the steam is allowed to escape through the tube a b c d, and the evaporation is continued until the contents of the flask are evaporated to about ten cubic centimeters. The flask into which the tube c d dips, is filled with freshly boiled water. The lamp is removed from the flask A, the pinch-cock is closed, whereupon the tube c d becomes filled with the freshly boiled water. The measuring tube C, filled with freshly boiled soda-lye is closed with the thumb and brought into the dish B, care being taken that no bubble of air enters. It is placed over the end of the tube g h.
The pressure of the external air will now flatten the rubber tubes at c and g. The flask at the end of c d holding freshly boiled water is then replaced with one filled with a nearly saturated solution of ferrous chlorid containing some hydrochloric acid. The flask containing the ferrous chlorid solution should be graduated so that the amount which is sucked into the flask A can be determined. The pinch-cock c is opened and from fifteen to twenty cubic centimeters of the ferrous chlorid solution allowed to flow into A. The end of the tube c d is then placed in another flask containing strong hydrochloric acid, and the latter allowed to flow into the tube in small quantities at a time until all the ferrous chlorid is washed out of the tube b c d into A. At the point b there is sometimes formed a little bubble of hydrochloric acid in the state of gas, which by heating the flask A completely disappears.
The flask A is next warmed gently until the rubber tubes at the pinch-cocks begin to assume their normal condition. The pinch-cock at g is now replaced by the thumb and finger, and as soon as the pressure within the flask A is somewhat stronger, caused by the nitric oxid gas evolved from the mixture, it is allowed to pass through the tube e f g h and escape into the measuring cylinder C. By a manipulation of the finger and thumb at g, it is possible to prevent regurgitation of the sodium hydroxid into A, and at the same time to relieve the pressure of the nitric oxid in A, which would be difficult to do by means of the pinch-cock alone.
The boiling of the liquid is continued until there is no longer any increase of the volume of gas in the measuring cylinder C. After the end of the operation the tube g h is removed from the dish B and the measuring tube C is closed by means of the thumb while its end is still beneath the surface of the soda-lye, and it is shaken until all traces of any hydrochloric acid, which may have escaped absorption, are removed. It is then placed in a large glass cylinder filled with water at the temperature at which the volume of gas is to be read. After being kept at this constant temperature for about half an hour the volume of the nitric oxid can be read. For this purpose the measuring cylinder C is sunk into the water of the large cylinder until the level of the liquids within and without the tube is the same. The usual correction for pressure of the atmosphere, as determined by the barometer, and for the tension of the aqueous vapor at the temperature at which the reading is made, is applied. The correction is made by means of the following formula:
V′ = (V × 273 × (B − f)) ÷ ((273 + t) × 760)
In this formula V′ denotes the volume of the gas at the temperature of zero, and at 760 millimeters barometric pressure; V the volume of the gas as read at the barometric pressure observed, B, and the temperature observed, t, while f denotes the tension of the aqueous vapor in millimeters of mercury pressure at the observed temperature t. The tension of the aqueous vapor at temperatures from zero to 26°, expressed in millimeters of mercury, is given in the following table:
─────┬───────────── Temp.│ Tension in ° │mm. mercury. ─────┼───────────── 0│ 4.6 1│ 4.9 2│ 5.3 3│ 5.7 4│ 6.1 5│ 6.5 6│ 6.9 7│ 7.4 8│ 8.0 9│ 8.5 10│ 9.1 11│ 9.7 12│ 10.4 13│ 11.1 14│ 11.9 15│ 12.7 16│ 13.5 17│ 14.4 18│ 15.3 19│ 16.3 20│ 17.4 21│ 18.5 22│ 19.6 23│ 20.9 24│ 22.2 25│ 23.5 26│ 25.0 ─────┴─────────────
From the gas volume reduced by the above formula the nitric acid is calculated as follows:
One cubic centimeter of nitric oxid weighs at 0° and 760 millimeters barometric pressure 1.343 milligrams.
Since two molecules of NO (molecular weight sixty) correspond to one molecule of N₂O₅ (108) we have the following equation: 60 : 108 = 1.343 : x. Whence x = 2.417 milligrams, the weight of nitric acid corresponding to one cubic centimeter of nitric oxid.
=467. DeKonick’s Modification of Schloesing’s Method.=—This modification consists in an arrangement of the gas delivery-tube, whereby the regurgitation of the water in the measuring burette into the evolution flask is prevented by a device for sealing the delivery-tube with mercury.[299] The apparatus is arranged as shown in Fig. 79. The flask in which the decomposition takes place is provided with a long neck, into which a side tube is sealed and bent upwards, carrying a small funnel attached to it by rubber tubing. The piece of rubber tubing carries a pinch-cock, by means of which the solution containing the nitrate and hydrochloric acid can be introduced into the flask. The small gas delivery-tube is arranged as shown in the figure, and carries at the end next the burette a device shown in Fig. 80. The cork represented in this device has radial notches cut in it, so as to permit of a free communication between the water in the burette and in the pneumatic trough. The open end of the burette, when the apparatus is mounted ready for use, rests on the notched surface of the cork, and the end of the delivery-tube is placed in the crystallizing dish resting on the bottom of the pneumatic trough.
The end of the delivery-tube, as indicated, has fused onto it a vertical tube open at both ends and six to seven centimeters in length, and carrying the notched cork already described. The crystallizing dish in the bottom of the pneumatic trough is filled with mercury until the point of union of the delivery-tube with the vertical end is sealed to the depth of a few millimeters. As the gas is evolved it bubbles up through the mercury into the measuring tube and the displaced water passes out through the notches in the cork. Should any back pressure supervene the mercury at once rises in the delivery-tube which is of such a length as to prevent its entrance into the flask. The operation can then be carried on with absolute safety.
To make an estimation there are placed in the flask about forty cubic centimeters of ferrous chlorid solution containing about 200 grams of iron to the liter, and also an equal volume of hydrochloric acid of one and one-tenth specific gravity. The side tube is also filled up to the funnel with the acid. The contents of the flask are boiled until all air is expelled, which can be determined by holding a test-tube filled with water over the end of the delivery-tube. The solution containing the nitrate is next placed in the funnel, the pinch-cock opened and the liquid allowed to run into the flask by means of the partial vacuum produced by stopping the boiling and allowing the mercury to rise in the delivery-tube. All the solution is washed into the flask by successive rinsings of the funnel with hydrochloric acid, being careful to allow no bubble of air to enter. The contents of the flask are again raised to the boiling-point and the nitric oxid evolved collected in the nitrometer. The solution examined should contain enough nitrate to afford from sixty to eighty cubic centimeters of gas. Without refilling the flask, from eight to nine determinations can be made by regenerating the ferrous chlorid by treatment with zinc chlorid. Care must be exercised not to add the zinc chlorid in excess, otherwise ammonia and not nitric oxid will be produced. The side tube and funnel must also be carefully freed from zinc chlorid by washing with hydrochloric acid.
=468. Schmidt’s Process.=—In the case of a water, or the aqueous extract of a soil, according to the content of nitric acid, from fifty to one hundred cubic centimeters are evaporated to thirty cubic centimeters, and the residue sucked into the generating flask of the apparatus, Fig. 81, and, with the rinsings with distilled water, evaporated again to from twenty to thirty cubic centimeters, and the flask then connected, as shown in the figure, to a Schliff measuring apparatus, B.[300] This apparatus is previously filled to i with mercury, and the bulb g connected with k by a rubber tube.
The apparatus is then filled with a twenty per cent, previously boiled and still warm, caustic soda solution until the bulb g is partially filled when raised a little above the cock h. Then h is closed and g held, by an appropriate support, on about the same level with h. The cock at b is then closed and e opened. Meanwhile the ebullition in the flask is continued, and the air bubbles rising in the Schliff apparatus are removed, from time to time, by carefully opening h and raising g. When bubbles no longer come over, the cock at e is closed and at b opened, and the steam issuing at a is conducted through a mixture of ferrous chlorid and strong hydrochloric acid to free it, as far as possible, from air. When the contents of the flask have been evaporated to about five cubic centimeters, b is closed and the lamp at once removed.
By carefully opening b about ten cubic centimeters of a mixture of ferrous chlorid and hydrochloric acid are allowed to enter the flask, when b is closed and the flask slowly heated until the positive pressure is restored. The pinch-cock e is then opened and the contents of the flask evaporated nearly to dryness. The cock e is again closed and the flame removed. Another quantity (fifteen cubic centimeters) of ferrous chlorid and hydrochloric acid solution is sucked into the flask and the process of distillation repeated, whereby the whole of the nitric oxid is collected in h. The nitric oxid evolved is measured in the usual way and calculated to nitric acid, one cubic centimeter of nitrogen dioxid being equal to 2.417 milligrams of nitric acid.
=469. Merits of the Ferrous Chlorid Process.=—The possibility of an accurate determination of nitrates; by decomposition with a ferrous salt in presence of an excess of acid, has been established by many years of experience and by the testimony of many analysts. The method is applicable especially where the quantity of nitrate is not too small and when organic matter is present. In the case of minute quantities of nitrate, however, the process is inapplicable and must give way to some of the colorimetric methods to be hereafter described.
In respect of the apparatus modern practice has led to the preference of that form which does not require the use of carbon dioxid for displacing the air. Steam appears to be quite as effective as carbon dioxid and is much more easily employed. That form of apparatus should be used which is the simplest in construction and has the least cubical content.
The measurement of the evolved gas is most simply made by collecting over lye in an azotometer, reading the volume, noting the reading of the barometer and thermometer and then reducing to standard conditions of pressure and temperature by the customary calculations. Where a very strong lye is used the tension of the aqueous vapor may be neglected. While every analyst should have a thorough knowledge of the ferrous chlorid method and the principles on which it is based it can not be compared in simplicity to the later methods with pure nitrates which are based on the conversion of the nitric acid into ammonia by the action of nascent hydrogen. In accuracy, moreover, it does not appear to have any marked advantage over the reduction methods.
=470. Mercury and Sulfuric Acid Method.=—This simple and accurate method of determining nitric acid in the absence of organic matter is known as the Crum-Frankland process.[301]
The method rests on the principle of converting nitric acid into nitric oxid by the action of mercury in the presence of sulfuric acid. The operation as at first described is conducted in a glass jar eight inches long by one and a half inches in diameter filled with mercury and inverted in a trough containing the same liquid. The nitrate to be examined, in a solid form, is passed into the tube together with three cubic centimeters of water and five of sulfuric acid. With occasional shaking, two hours are allowed for the disengagement of the gas, which is then measured.
=471. Warington’s Modification.=—A graduated shaking tube is employed which allows the nitrate solution and oil of vitriol to be brought to a definite volume. The nitrate solution, with rinsings, is always two cubic centimeters and enough sulfuric acid is added to increase the volume to five cubic centimeters. The sulfuric acid should give no gas when shaken with distilled water. Any gas given off in the apparatus before shaking, is not expelled but is included in the final result. The persistent froth sometimes noticed where some kinds of organic matter are present, is reduced by the addition of a few drops of hot water through the stop-cock of the apparatus. The nitric oxid is finally measured in Frankland’s modification of Regnault’s apparatus.
This method, accurate for pure nitrates, unfortunately fails in the presence of any considerable amount of organic matter.
According to Warington’s observations the presence of chlorids is no hindrance to the accurate determination of both nitric and nitrous acids by the mercury method. This simplifies the operation as carried on by Frankland who directs that any chlorin present, be removed before the determination of the nitric acid is commenced.
=472. Noyes’ Method.=—In the analyses made by Noyes for the National Board of Health, the Crum-Frankland method was employed.[302] The apparatus used was essentially that which is now known as Lunge’s nitrometer and it will be described in the next paragraph. No correction is made by Noyes for the tension of aqueous vapor in the measurement of the nitric oxid because of the moderate dilution of the sulfuric acid by the liquid holding the nitric compounds in solution. The chlorin was not removed from the dry residue of the evaporated water as its presence in moderate quantity does not interfere with the accuracy of the process. In order to obtain the amount of nitrogen in the form of nitrates, the total volume of nitric oxid must be diminished by that due to nitrites present, which must be determined in a separate analysis. The method of manipulation is given in the following paragraph.
=473. Lunge’s Nitrometer.=—The apparatus employed by Noyes, in a somewhat more elaborate form, is known as Lunge’s nitrometer.[303] This apparatus is shown in Fig. 82. It consists of a burette, a, divided into one-fifth cubic centimeters. At its upper end it is expanded into a cup-shaped funnel attached by a three-way glass stop-cock. Below, the burette is joined to a plain tube, b, of similar size, by means of rubber tubing. The apparatus is first filled with mercury through the tube b, the stop-cock being so adjusted as to allow the mercury to fill the cup at the top of a. The cock is then turned until the mercury in the cup flows out through the side tube carrying the rubber tube and clamp. The three-way cock is closed, and the solution containing the nitrate placed in the cup. By lowering the tube b and opening the cock the liquid is carefully passed into a, being careful to close the cock before all the liquid has passed out of the cup. By repeated rinsings with pure concentrated sulfuric acid, every particle of the nitric compound is finally introduced into a, together with a large excess of sulfuric acid. The total volume of the introduced liquid should not exceed ten cubic centimeters. The mixture of the mercury, nitric compound, and sulfuric acid is effected by detaching a from its support, compressing the rubber connection between a and b, placing a nearly in a horizontal position, and quickly bringing it into a vertical position with vigorous shaking.
After about five minutes the reaction is complete, and the level of the liquids in the two tubes is so adjusted as to compensate for the difference in specific gravity between the acid mixture in a and the mercury in b; in other words, the mercury column in b should stand above the mercury column in a one-seventh of the length of the acid mixture in a. This secures atmospheric pressure on the nitric oxid which has been collected in a. The measured volume of nitric oxid should be reduced to 0° and 760 millimeters barometric pressure. Each cubic centimeter of nitric oxid thus obtained corresponds to 1.343 milligrams NO; 2.417 milligrams N₂O₅; 4.521 milligrams KNO₃; 1.701 milligrams N₂O₃; 2.820 milligrams HNO₃; and 3.805 milligrams NaNO₃.
=474. Lunge’s Improved Apparatus.=—Lunge has lately improved his apparatus for generating and measuring gases and extended its applicability.[304] The part of it designed to measure the volume of a gas is the same in all cases. For generating the gas, the apparatus varies according to the character of the substance under examination.
The measuring apparatus is shown in Fig. 83. It is composed essentially of three tubes, conveniently mounted on a wooden holder with a box base for securing any spilled mercury. The support is not shown in the illustration.
The tubes A, B, C, are mutually connected by means of a three-way tube and rubber tubing with very thick walls to safely hold the mercury without expansion. In the middle of the measuring tube A, is found a bulb of seventy cubic centimeters capacity. Above and below the bulb the tube is divided into tenths of a cubic centimeter, and its diameter is such, viz., 11.3 millimeters, that each cubic centimeter occupies a length of one centimeter. The upper end of A is closed with a glass cock with two oblique perforations, by means of which communication can be established at will, either through e with the apparatus for generating the gas, or through d with the absorption apparatus, or the opening be completely closed.
The volume of air under the observed conditions which would measure exactly 100 cubic centimeters at 0° and 760 millimeters pressure of mercury, is calculated by the formula
V = (100(273 + t)760)/(273(b − f));
where t equals observed temperature, b the barometric pressure less the correction noted above and f the tension of the vapor of water under existing conditions. For example:
Let the temperature be 18° Barometric reading 755 Correction for t 2 Corrected barometer 753 Vapor of water tension 16
Then V = (100(273 + 18)760)/(273(753 − 16)) = 109.9.
This indicates that 109.9 cubic centimeters of air would occupy a volume of 100 cubic centimeters when subjected to standard conditions.
The tubes A, B, and C are filled with mercury of which about two and a half kilograms will be required. By means of the leveling tube B, the stopper in C being opened, the mercury in C is brought exactly to 109.9 cubic centimeters. The stopper in C is then closed, mercury poured into D, which is then closed with a rubber stopper, carrying a small glass tube as indicated in the figure.
The leveling tube B serves to regulate the pressure on the gas in A and this is secured by depressing or elevating it as the case may require.
The tube for reducing the volume to standard conditions of temperature and pressure, viz., 0° and 760 millimeters of mercury, is shown in C. In its narrow part which has the same internal diameter as A it is graduated into tenths of a cubic centimeter. The upper end of C is furnished with a heavy glass neck D surmounted by a glass cup. In the neck is placed a ground-glass stopper, carrying a groove below, which corresponds to a similar groove above in the side of the neck whereby communication can be established at will between the interior of C and the exterior. The joint is also sealed by pouring mercury into D as is shown in the figure. When the stopper is well ground and greased the reduction tube may be raised or lowered as much as may be necessary without any danger of escape or entrance of gas. To determine the position of the reduction tube C the reading of the barometer and thermometer at room temperature is taken. From the reading of the barometer subtract one millimeter if the temperature be below 12°, two millimeters at a temperature from 12° to 19°, three from 20° to 25°, and four above 25°.
When a gas has been introduced into the measuring tube A it is brought to the volume which it would assume under standard conditions by adjusting the tube C in such a way as to bring the level of mercury in C and A to the same point and the level of the mercury in C is exactly at 100 cubic centimeters. The gas in A is then at the volume which it would occupy under standard conditions and this volume can be directly read. This adjustment is secured by moving the tubes B and C up or down. If gases are to be measured wet, a drop of water should be put on the side of the upper part of C, and, if dry, of sulfuric acid, before the adjustment for temperature and pressure.
=475. Method of Manipulation.=—By the action of mercury in the presence of sulfuric acid, the nitrogen in nitrates, nitrites, nitrosulfates, nitroses, nitrocellulose, nitroglycerol, and the greater number of explosives, may be obtained and measured as nitric oxid. The nitrogen compounds are decomposed in the apparatus shown in Fig. 84.
To make an analysis, the apparatus is filled with mercury, through F, until the two openings in the cock and i are entirely occupied with that liquid. The cock h is then closed, and the nitrogen compound, in solution, introduced through g, care being taken that no air enters g when F is depressed and h opened to admit the sample. The funnel g is washed several times with a few drops of sulfuric acid, which are successively introduced into G. The total liquid introduced should not exceed ten to fifteen cubic centimeters, of which the greater part should be sulfuric acid. The rubber tube connecting G and F is carefully closed with a clamp and G violently shaken for a few minutes until no further evolution of nitric oxid takes place. In shaking, the apparatus should be so held as to prevent the escape of the mercury from the small tube i by keeping it closed with the finger or drawing over it a rubber cap.
After the evolution of the gas has ceased, the tube e, Fig. 83, is brought into contact with i, Fig. 84, and the two are joined by a tight-fitting piece of rubber tubing in such a way as to exclude any particle of air. The tube F, Fig. 84, is lifted and B and C, Fig. 83, depressed. On carefully opening the cocks h and b and bringing i and e into union, the gas is passed from G into A. When all the gas has entered A and the acid mixture from G has reached b the latter is closed, and also h. The apparatus G is disconnected and removed. The gas in A is then reduced to normal conditions by manipulating the reduction tube C in the manner already described.
The gas in A is measured dry by reason of having been generated in presence of rather strong sulfuric acid. Consequently, for this operation the adjustment of the volume of gas in C should be made in contact with a drop of strong sulfuric acid. In order to make the readings, a quantity of material must be taken which will give less than thirty or from 100 to 140 cubic centimeters of nitric oxid.
The quantities of the different compounds of nitric acid corresponding to the number of cubic centimeters of nitric oxid, measured under standard conditions, are shown in the following table:
CORRESPONDING TO ———————————— ———————————— ———————————— Cubic Weight in N₂O₃ in HNO₃ in NaNO₃ in centimeters milligrams. milligrams. milligrams. milligrams. of NO. 1 1.343 1.701 2.820 3.805 2 2.682 3.402 5.640 7.610 3 4.029 5.103 8.460 11.415 4 5.372 6.804 11.280 15.220 5 6.715 8.506 14.100 19.025 6 8.058 10.206 16.920 22.830 7 9.401 11.907 19.740 26.635 8 10.744 13.608 22.560 30.440 9 12.087 15.309 25.380 34.245
=476. Utility of the Method.=—Where it is desirable that the nitric oxid method be used, and at the same time heating be avoided, the decomposition of a nitrate by means of metallic mercury and sulfuric acid affords a convenient and accurate procedure. But, as a rule, there is no objection to the application of the lamp, and in such cases the mercury method appears to have no advantage over the ferrous chlorid process. Nevertheless, in the hands of a skilled worker the results are reliable, and the process is a quicker one, on the whole, than by distillation with ferrous chlorid and hydrochloric acid. This method, however, can not be recommended as in any way superior to the reduction methods to be hereinafter described.
ESTIMATION OF NITRIC ACID BY OXIDATION OF A COLORED SOLUTION.
=477. Method of Boussingault.=—The process for the estimation of nitric acid by the decoloration of a solution of indigo is due originally to Boussingault.[305] In this method the extract, obtained by washing slowly 200 grams of soil until the filtrate amounts to 300 cubic centimeters, is evaporated until its volume is no greater than two or three cubic centimeters, and it is transferred to a test-tube, with washings, and again evaporated in the tube until the volume is not greater than that last mentioned. A few drops of solution of indigo are added, and then two cubic centimeters of pure hydrochloric acid; the whole is then heated. As the color of the indigo disappears more is added. When the color ceases to fade, the liquid in the test-tube is concentrated by boiling. If concentration fail to destroy the blue or green color, another one-half cubic centimeter of hydrochloric acid is introduced. The reaction is completed when neither concentration nor fresh addition of hydrochloric acid destroys the excess of indigo present. The color produced by a small excess of indigo is a bright sap-green; this tint is the final reaction sought. The small excess of indigo necessary to produce a green color is deducted in every experiment.
When more than mere traces of organic matter are present, Boussingault advises that the nitric acid be first separated by distillation and then reduced by the indigo solution. For this purpose the concentrated solution of the nitrate, two or three cubic centimeters, is placed in a small tubulated retort with two grams of manganese dioxid in fine powder. The retort is next half filled with fragments of broken glass, over which is poured one cubic centimeter of concentrated sulfuric acid. The retort is heated carefully by means of a small flame, which is kept in motion so as to successively come in contact with all parts of the bottom of the retort. The distillate is received in a graduated test-tube which is kept cool. The distillation is continued until the vapors of sulfuric acid begin to appear. The apparatus is allowed to cool, the stopper of the retort removed, two cubic centimeters of water introduced, and the distillation again made until fumes of sulfuric acid are again seen. The distillation with water is made twice in order to remove every trace of nitric acid from the retort. The distillate is neutralized with a solution of potassium hydroxid and concentrated to two cubic centimeters, and the nitric acid estimated in the manner already described. The manganese dioxid used should be previously well washed and the sulfuric must be free of nitric acid.
Preparation of the Indigo Solution.—Fifty grams of indigo in fine powder are digested for twenty-four hours, at 40°, in a liter of distilled water. The water is then poured off and replaced with a fresh supply. After the second decantation the residue is treated with 750 cubic centimeters of equal parts of water and pure concentrated hydrochloric acid and boiled for an hour. After cooling, the undissolved portion is collected on a filter and washed at first with hot, and afterwards with cold water, until the filtrate is no longer colored and is free of acid. The dried residue is treated with ether under a bell-jar, or in a continuous extraction apparatus, until the ether is only of a faint blue tint. The fifty grams of indigo at first taken will give about twenty-five grams of the purified article, which, however, will still leave a little ash on combustion.
Solution in Sulfuric Acid.—Five grams of the purified indigo are placed in a flask having a ground-glass stopper, treated with twenty-five grams of fuming sulfuric acid, and allowed to digest two or three days at a temperature of from 50° to 60°. From seventy to 200 drops of the solution thus made are placed in 100 cubic centimeters of water for use in the process.
Standardization of the Indigo Solution.—The solution as prepared above is standardized by a solution of one gram of pure potassium nitrate in 1,000 cubic centimeters of distilled water. The oxidation of the indigo solution is accomplished as described above. For this strength of standard nitrate solution two cubic centimeters are taken corresponding to two milligrams of potassium nitrate. The indigo solution for this strength should have only twenty drops of the sulfuric acid solution of indigo to 100 cubic centimeters of water. If twenty grams of potassium nitrate are taken for 1,000 cubic centimeters of the standard solution then 200 drops of the sulfindigotic acid should be used to 100 cubic centimeters of water.
=478. Method of Marx.=—As usually practiced, the indigo method is conducted according to the variation described by Marx.[306] There are required for the process the following reagents and apparatus:
a. A solution of pure potassium nitrate containing 1.8724 grams per liter. One cubic centimeter of the solution is equivalent to one milligram of nitric anhydrid (N₂O₅).
b. A solution of the best indigo carmine in water which should be approximately standardized by solution in the manner described hereafter, and then diluted so that six to eight cubic centimeters equal one milligram of nitric acid.
c. Chemically pure sulfuric acid of specific gravity 1.842, perfectly free from sulfurous and arsenious acids and nitrogen oxids.
d. Several thin flasks of about 200 cubic centimeters capacity.
e. A small cylindrical measure holding fifty cubic centimeters and divided into cubic centimeters.
f. A Mohr’s burette divided into tenths of a cubic centimeter.
g. A twenty-five cubic centimeter pipette or another burette.
h. A five cubic centimeter pipette divided into cubic centimeters or half cubic centimeters.
i. A measuring flask of 250 cubic centimeters capacity.
Preliminary Trial.—Twenty-five cubic centimeters of the sample are transferred to a flask; the fifty cubic centimeter measure is filled with sulfuric acid and the burette with indigo solution. The sulfuric acid is added to the sample all at once, shaken for a moment, and the indigo run in as quickly as possible with shaking until a permanent greenish tint is produced. If the sample do not require more than twenty cubic centimeters of indigo solution of the above strength, it can be titrated directly, otherwise it must be diluted with a proper quantity of pure water, and subjected again to the preliminary trial.
The Actual Titration.—(1) Twenty-five cubic centimeters of the sample properly diluted if necessary, are measured and poured into a flask, and as much indigo as was used in the preliminary trial, is added; a quantity of sulfuric acid, equal in volume to the liquid in the flask, is added all at once, the mixture shaken, and indigo solution run in quickly out of the burette until the liquid remains permanently of a greenish tint.
(2) The last experiment is repeated as often as may be necessary adding to the water at first half a cubic centimeter less indigo than the total quantity used previously, afterwards proceeding as in (1) until the final test shows too little indigo used.
(3) From the rough titration of the indigo, calculate the amount of potassium nitrate solution corresponding with the indigo solution used in (2), multiply the result by ten, transfer this quantity of the standard nitrate solution to a 250 cubic centimeter flask, fill with pure water to the mark, and titrate twenty-five cubic centimeters of this fluid with indigo as in (2). If the quantity of indigo solution used is nearly the same as that required in (2), its exact value may be calculated, but if it is not, another nitrate solution may be made up in the 250 cubic centimeter flask, more closely resembling the sample in strength, and the titration with the indigo solution must be repeated.
(4) If the water contain any considerable amount of organic matter, it must first be destroyed by potassium permanganate. In this case, the estimation of the organic matter and nitric acid may be conveniently combined.
The use of permanganate in the above case is likely to introduce an error as has been shown by Warington. The method therefore can not be recommended in the presence of organic matter.
=479. Method of Warington.=—The modification of the indigo method as used by Warington, applicable only in absence of organic matter, is the one chiefly employed in England.[307]
Instead of the ordinary indigo of commerce, indigotin is used. The normal solution of the coloring matter is made of such a strength as to be equivalent to a solution of potassium nitrate containing 0.14 gram of nitrogen per liter. Where large quantities of the coloring matter are to be used it is advisable to prepare it about four times the strength given above and then dilute it as required. Four grams of sublimed indigotin will furnish more than two liters of the color solution.
The solution is prepared as follows:
Four grams of indigotin are digested for a few hours with five times that weight of Nordhausen sulfuric acid, diluted with water, filtered, and made up to a volume of two liters. The strength of the indigotin solution is determined with a solution of potassium nitrate of the strength mentioned above. The process is performed as follows:
From ten to twenty cubic centimeters of the standard nitrate solution are placed in a wide-mouthed flask of about 150 cubic centimeters capacity. A portion of the indigotin solution is next added, such as will be deemed sufficient for the process, and the whole is well mixed. Strong sulfuric acid is next measured out from a burette into a test-tube, in volume equal to the united volumes of the nitrate solution and indigotin. The whole of the sulfuric acid is then poured as quickly as possible, into the solution in the flask and rapidly mixed, and the flask transferred to a calcium chlorid bath, the temperature of which should be maintained at 140°. It is essential to the success of the operation that the sulfuric acid should be mixed with the greatest rapidity. It should be poured in at once and the whole well shaken without waiting for the test-tube containing the acid, to drain. The flask should be covered by a watch-glass while it is held in the bath. As soon as the larger part of the indigotin is oxidized the flask in the bath should be gently rotated. With very weak solutions of nitrate it may be necessary sometimes to keep the flask in the bath for five minutes. When the indigo color is quickly discharged it shows the presence of nitric acid in considerable excess and a considerably larger quantity of indigo must be taken in the next experiment. The experiments are continued until just the quantity of indigo necessary to consume the nitric acid is taken, the amount of indigo being in very slight excess, not exceeding one-tenth cubic centimeter of the indigo solution used. The tint produced by the small excess of indigo remaining is best seen by filling the flask with water. On substances of approximately known strength about four experiments are usually necessary to determine the amount of indigo to be taken, but with unknown substances a larger number may be necessary.
Usually in determinations of this kind it is directed to use double the volume of sulfuric acid mentioned above. In this case not only is the quantity of indigo oxidized much greater than with a smaller portion of acid, but the prejudicial effect of organic matter is also greater than when the smaller quantity of acid is employed.
An indigo solution standardized as above is strictly to be used for a solution of nitrate of the strength employed during the standardization. The quantity of indigo oxidized in proportion to the nitric acid present diminishes as the nitrate solution becomes more dilute. Instead of determining this during each series of experiments it may be estimated once for all and a table of corrections used.
The following table is based upon experimental determinations:
Strength of Indigo Difference Nitrogen Difference Difference niter required, between corresponding between the in the solution cubic amounts of to one cubic nitrogen nitrogen used. centi- indigo, centimeter of values, values for a meters. cubic indigo, gram. gram. difference centi- of one cubic meters. centi- meter in the amount of indigo, gram. ⁸⁄₆₄ Normal 10.00 0.000035000 ⁷⁄₆₄ „ 8.71 1.29 0.000035161 0.000000161 0.000000125 ⁶⁄₆₄ „ 7.43 1.28 0.000035330 0.000000169 0.000000132 ⁵⁄₆₄ „ 6.14 1.29 0.000035627 0.000000298 0.000000231 ⁴⁄₆₄ „ 4.86 1.28 0.000036008 0.000000381 0.000000298 ³⁄₆₄ „ 3.57 1.29 0.000036763 0.000000756 0.000000586 ²⁄₆₄ „ 2.29 1.28 0.000038209 0.000001445 0.000001129 ¹⁄₆₄ „ 1.00 1.29 0.000043750 0.000005541 0.000004295
The table is used as follows:
Suppose that twenty cubic centimeters of water under examination have required 5.36 cubic centimeters of indigo solution for the oxidation of the nitric acid contained therein. By inspection of the table it is seen that this number is five-tenths cubic centimeter above the nearest quantity given; viz., 4.86 cubic centimeters. From the last column in the table it is found that the correction for five-tenths cubic centimeter of indigo solution is 0.000000149 cubic centimeter, being half that for the one cubic centimeter given in the table. This is to be subtracted from the unit value in nitrogen given in the first “gram” column of the table; viz., 0.000036008. It is thus seen that the 5.86 cubic centimeters of indigo solution are equivalent to 0.000035859 gram of nitrogen per cubic centimeter. The water under examination, therefore, contains nine and six-tenths parts of nitrogen as nitric acid per million.
Attention must also be paid in standardizing indigo solutions to the initial temperature of the solutions. A rise in the initial temperature will be attended by a diminution in the quantity of indigo oxidized. Experiments with a room temperature of 10° and a room temperature of 20°, being the initial temperatures of the experiments, showed that at the higher temperature the amount of indigo consumed was about five per cent less when the strong solutions of nitrate were employed. The indigo solution should, therefore, be standardized at the same temperature at which the determinations are made.
If twenty cubic centimeters of the standard nitrate solution employed be used in setting the indigo solution, this standard will enable the operator to determine nitric acid up to 17.5 parts of nitrogen per million in water or soil extracts.
The presence of an abundance of chlorids in the water under examination tends to diminish the content of nitric acid found, and also tends to introduce an error, which is sometimes of a plus and sometimes of a minus quantity, according to the strength of the nitric acid present. The reaction is shortened in weak solutions by the presence of chlorids, and the quantity of indigo consumed is consequently increased. The error introduced by chlorids is usually of an insignificant nature.
On account of the interference of organic matters with the reaction of indigo it is not of much use in the examination of nitrates washed out of soils, although in some cases the results may be quite accurate. This method must, therefore, be considered as applicable, in general, to waters or soil extracts which contain little or no organic matter.
In analytical work pertaining particularly to agriculture, the use of the indigo method for determining nitric acid has been largely employed, both in the analyses of soil extracts and drainage and irrigation waters. The method, however, can hardly survive as an important one in such work in competition with more modern and speedy processes of analysis.
DETERMINATION OF NITRIC NITROGEN BY REDUCTION TO AMMONIA.
=480. Classification of Methods.=—When nitrogen is present in a highly oxidized state, e. g., as nitric acid, it may be quickly and accurately estimated by reduction to ammonia. This action is effected by the reducing power of nascent hydrogen, and this substance may be secured in the active state by the action of an acid or alkali on a metal, or by means of an electric current. The processes depending on the use of a finely divided metal in the presence of an acid or alkali have come into general use within a few years, and are now employed generally instead of the more elaborate estimations depending on the use of copper oxid or indigo.
The typical reaction which takes place in all cases is represented by the following equation:
2HNO₃ + 8H₂ = 2NH₃ + 6H₂O.
The method will be considered under three heads; viz., 1. Reduction in an alkaline solution; 2. Reduction in an acid solution; 3. Reduction by means of an electric current.
In the first class of processes the reduction and distillation may go on together. In the second class the reduction is accomplished first and the distillation effected afterwards, with the addition of an alkali. In the third class of operations the reduction is accomplished by means of an electric current and the ammonia subsequently obtained by distillation, or determined by nesslerizing. These processes may be applied to rain and drainage waters, and to soil extracts. On account of the ease with which the analyses are accomplished, the short time required and the accuracy of the results, the reduction methods for nitrates have already commended themselves to analysts, and are quite likely to supersede all others for practical use where weighable quantities of nitrates are present. For the minute traces of nitrates found in rain and drainage waters, and in some soil extracts, the reduction method may also be applied, but in these cases the ammonia which is formed must be determined by colorimetry (nesslerizing) and not by distillation. The processes about to be described are especially applicable to the examination of soils and waters rich in nitrates.
REDUCTION IN ALKALINE SOLUTIONS.
=481. Provisional Method of the Association of Official Agricultural Chemists.=[308]—
Extraction of the Nitrates.—Place one kilogram of the dried soil, calculated to water-free substance, on a percolator of glass or tin. Moisten the soil thoroughly with pure distilled water, and allow to stand for half an hour. Add fresh portions of pure distilled water until the filtrate secured amounts to one liter. If the first filtrate be cloudy before use it may be refiltered.
Qualitative Test for Nitrates.—Evaporate five cubic centimeters of the soil extract in a porcelain crucible, having first dissolved a small quantity of pure brucin sulfate therein. When dry, add to the residue a drop of concentrated sulfuric acid free of nitrates. If the nitrate calculated as potassium nitrate does not exceed the two-thousandth part of a milligram only a pink color will be developed; with the three-thousandth part of a milligram a pink color with reddish lines; with the four-thousandth part of a milligram a reddish color; with the five-thousandth part of a milligram a distinct red color.
Estimation of the Nitrates.—Evaporate 100 cubic centimeters of the soil extract to dryness on a steam-bath. Dissolve the soluble portions of the residue in 100 cubic centimeters of ammonia-free distilled water, filtering out any insoluble residue. Place the solution in a flask, add ten cubic centimeters of sodium amalgam, stopper the flask with a valve which will permit the escape of hydrogen, and allow to stand in a cool room for twenty-four hours. Add fifty cubic centimeters of milk of lime and titrate the ammonia produced by distillation, with standard acid and estimate as nitrogen pentoxid. Where the amount of ammonia is small, nesslerizing may be substituted for titration.
Preparation of Sodium Amalgam.—Place 100 cubic centimeters of mercury in a flask of half a liter capacity; warm until paraffin will remain melted over the surface; drop successively in the paraffin-covered mercury, pieces of metallic sodium of the size of a pea until 6.75 grams have united with the mercury. The amalgam contains then 0.5 per cent of metallic sodium and may be preserved indefinitely under the covering of paraffin.
=482. Method of the Experiment Station at Möckern.=[309]—The principle of this reaction is based on the reducing action exercised by nascent hydrogen on a nitrate, the hydrogen being generated by the action of soda-lye on a mixture of zinc dust and finely divided iron.
Ten grams of nitrate are dissolved in 500 cubic centimeters of water. Of this solution twenty-five cubic centimeters, corresponding to one-half gram, are placed in a distillation flask of about 400 cubic centimeters capacity, 120 cubic centimeters of water added, and about five grams of well-washed and dried zinc dust and an equal weight of reduced iron. To the solution are added eighty cubic centimeters of soda-lye of 32° B. The flask is then connected with the condensing apparatus and the distillation carried on synchronously with the reduction, the ammonia being collected in twenty cubic centimeters of titrated sulfuric acid. The distillation is continued from one to two hours, or until 100 cubic centimeters have been distilled, and the remaining sulfuric acid is titrated in the usual way. Soil extracts and sewage waters should be concentrated until they have approximately the proportion of nitrates given above.
=483. Method of Devarda.=—The inconvenience due to slow action and other causes, arising from the use of pure metals in the reduction of nitrates to ammonia, has been overcome, to some extent, by Devarda, by use of an alloy, in a state of fine powder, consisting of aluminum, copper, and zinc.[310] The alloy consists of forty-five per cent of aluminum, fifty per cent of copper, and five per cent of zinc. In dissolving, the copper is left in a finely divided state, which is a great help in distillation in preventing bumping.
The analytical process is carried out as follows: The solution containing the nitrate, in quantity equivalent to about one-half gram of potassium nitrate, is placed in a flask having a capacity of about one liter, and diluted with sixty cubic centimeters of water and five cubic centimeters of alcohol, and then forty cubic centimeters of caustic potash solution added of specific gravity one and three-tenths. From two to two and one-half grams of the alloy, described above, are introduced, and the flask attached to a condenser with a receiver containing standard acid. The connection between the flask and the condenser is made by means of a tube having on the limb next the flask a bulb filled with glass beads to prevent the contents of the flask splashing over into the receiver, and on the other limb another bulb to prevent the acid in the receiver finding its way into the distillation flask, should regurgitation occur. When the flask has been thus connected with the condenser it is gently heated for half an hour, at the end of which time the evolution of hydrogen will have slackened or ceased, and then the distillation is begun, at first cautiously, until the zinc of the alloy has completely dissolved, and then more vigorously, the time necessary being about twenty minutes from the time when the contents of the flask begin to boil. The distillate is caught in standard acid and the ammonia determined by backward titration in the ordinary way. It is to be noted that the strength of the alkali used is of importance, as if it be too strong, the action on the alloy is unduly vigorous at the beginning of the operation, and if too weak, the contents of the flask have to be heated overmuch, the result in both cases being the formation of a fine spray of caustic solution, which is very difficult to stop, even with complicated washing attachments to the distilling flask. The test analyses on pure nitrates are satisfactory. This method has been used with satisfaction in the laboratory of the Department of Agriculture, but does not appear to have any special advantage over the process of Ulsch, to be described further on.
=484. Variation of Stoklassa.=—Stoklassa has subjected the method of Devarda to a comparative test with the following methods:[311]
1. Wagner’s Schloesing-Grandeau method.
2. Lunge’s nitrometer method.
3. Stutzer’s method.
The reduction takes place in a copper erlenmeyer, A, Fig. 85, in which, in addition to the solution containing the nitrate, are placed 200 cubic centimeters of water, forty cubic centimeters of potassium hydroxid solution of 33° B., five cubic centimeters of alcohol, and finally two and one-half grams of the finely powdered Devarda alloy. The distillate passes through a tube, B, filled with glass pearls and into the condenser D, through the bulbs, C C′.
After the flask is connected with the distilling apparatus, it is gently warmed and the reduction is ended in about twenty minutes. The ammonia which is formed is then distilled into E, containing the standard acid, S, requiring about twenty minutes more. The comparative results given, show that the Devarda method is equally as accurate as any of the other methods mentioned, giving practically theoretical results.
In so far, however, as speed of an analysis, is concerned the first place is awarded to the Lunge nitrometer method, with which a complete analysis can be made in from thirty to forty minutes. In the second rank, so far as speed is concerned, the Devarda method is recommended. All the methods give accurate results.
=485. Method of Sievert.=[312]—Two grams of potassium or sodium nitrate are dissolved and made up to 1,000 cubic centimeters. Fifty cubic centimeters of the solution are placed in a 600 cubic centimeter flask and diluted with fifty cubic centimeters of water, and from eighteen to twenty grams of caustic alkali added. After the alkali is dissolved, seventy-five cubic centimeters of ninety-six per cent alcohol are added and a few pieces of bone-black to prevent foaming. From ten to fifteen grams of zinc or iron dust are then added to the flask which is closed and connected with a =ᥩ= tube holding about 200 cubic centimeters, which contains about ten cubic centimeters of normal sulfuric acid. This =ᥩ= tube is kept cool by being immersed in water. The whole mixture is now allowed to stand for three or four hours and then the alcohol is distilled slowly and the ammonia formed by the reduction of the nitrates is carried over with it. The distillation lasts for about two hours. The contents of the =ᥩ= tube are carefully rinsed into a dish and the excess of sulfuric acid titrated with one-fourth normal soda-lye.
For soil extracts and substances containing unknown quantities of nitric acid, a preliminary test will indicate approximately the amount thereof, and this will be an indication for the quantity to be used in the analysis.
The method of Stutzer differs from the foregoing in the substitution of aluminum dust instead of iron or zinc.[313] The reducing power of aluminum, however, varies greatly according to the method in which the metal has been prepared. Pure aluminum prepared by the electric method, reduces the nitric acid much less vigorously than the metal prepared by the older methods of fusion with sodium. For this reason the method of Stutzer is not to be preferred to that of Sievert.
REDUCTION IN AN ACID SOLUTION.
=486. Variation of the Sodium Amalgam Process.=—This method is described by Monnier and Auriol.[314]
The principle of the operation depends on the reduction of the dissolved nitrate by titrated sodium amalgam in presence of an acid, and the estimation of the quantity of nitric acid present from the deficit in the volume of hydrogen. The apparatus employed is conveniently mounted as shown in Fig. 86. The brass vessel A is movable by means of the cord on the pulley B, in such a way as to be fixed at any required altitude. It is filled with water and connected by a rubber tube to the cooling tube D. Within the cooling tube there is a graduated cylinder open at its lower end. Its upper end is connected directly with the apparatus C. The cooling tube D has a small side tube, c, near its upper end, by means of which the air can enter or escape when the position of A is changed. The apparatus C, in which the reaction takes place, is a glass cylinder. Its upper end is continuous with the =⟙= tube provided with the stop-cocks a and b. One arm of the =⟙= permits connection with the graduated measuring tube by means of a rubber union. The lower end of C is closed with a large hollow ground-glass stopper, carrying a small receptacle within, so that it forms two separate water-tight compartments, open at the top.
The sodium amalgam is prepared as follows:
In a clay crucible are heated 400 grams of mercury, and, little by little, with constant stirring, four grams of dry sodium are added. When cold, the amalgam is placed in a burette, having a ground-glass stopper, and covered with petroleum. The strength of the amalgam is established in the following manner. A small glass thimble, ground even at the top, is filled with the amalgam and struck off even with a ground-glass straight edge. In this way the same quantity of amalgam is taken for each test. This measured portion of the amalgam is placed in the inner vessel of the glass stopper to C. Ten cubic centimeters of water, containing sixty centigrams of tartaric acid, are placed on the outer ring of the glass stopper, which is then inserted, well oiled, in C, closing it air- and water-tight. The tartaric acid solution also carries a piece of litmus paper, so that its constant acidity may be insured. The vessel A is then fixed in a position which brings the water in the graduated burette and tube D exactly to the 0 mark. The cock a is next closed, b opened, and C is inverted until all the amalgam is poured into the solution of tartaric acid. The evolved hydrogen mixed with the air contained in the apparatus, is passed into the graduated burette. After fifteen minutes, the reaction is ended. The water level within and without the graduated tube is restored and the volume of gas evolved noted and reduced by the usual tables to 0° and 760 millimeters pressure of the barometer.
An amalgam prepared as above will give about three cubic centimeters of hydrogen for each gram. The thimble should hold from twelve to fifteen grams.
The estimation of nitric acid should be made in a solution containing about one-tenth per cent of nitrate. Ten cubic centimeters are taken, to which six-tenths gram of tartaric acid is added, and placed in the outer part of the glass stopper. The rest of the process is conducted exactly as described above. The deficit in hydrogen is calculated to nitrogen pentoxid.
The reduction by sodium amalgam is not so convenient a form of estimating nitric acid as many of the other forms of using nascent hydrogen. As practiced by calculating from the deficit of hydrogen, however, it has some advantages by reason of the fact that no heating is required. The presence of organic neutral bodies, or even those of an acid nature, like humus, does not, therefore, interfere with the work. Likewise, mineral bodies in solution, which are not reduced by nascent hydrogen, do not interfere with the accuracy of the reaction.
=487. Method of Schmitt.=—In the method of Schmitt forty cubic centimeters of glacial acetic acid are placed in a flask of 600 cubic centimeters content, and fifteen grams of a mixture of zinc and iron dust added.[315] To this a quantity of the solution containing the nitrate, representing about half a gram of the pure nitrate, is added with constant shaking, in portions which do not evolve hydrogen too rapidly. After about fifteen minutes when the evolution of nitrogen has somewhat diminished, an additional fifteen grams of the metal dust are added. If the contents of the flask should become thick they can be diluted with thirty cubic centimeters of water. The reduction is complete in from thirty to forty minutes. The contents of the flask are now saturated with enough soda-lye not only to neutralize the excess of acetic acid, but to keep the zinc hydroxid also in solution. For this purpose about 200 cubic centimeters of soda-lye of 1.25 specific gravity are necessary. The ammonia is obtained by distillation into standard acid in the usual way.
=488. Method of Ulsch.=—In practice the method of Ulsch has come into general use.[316]
For the determination of nitrogen by this method half a gram of saltpeter or four-tenths gram of sodium nitrate is taken and dissolved in twenty five cubic centimeters of water, in a flask with a content of about 600 cubic centimeters. Five grams of iron reduced by hydrogen, and ten cubic centimeters of sulfuric acid diluted with two volumes of water are then added to the flask. To avoid mechanical losses during the evolution of hydrogen a pear-shaped glass stopper is hung in the neck of the flask. After the first violent evolution of hydrogen has passed, the flask is slowly heated until in about four minutes it is brought to a gentle boil. The boiling is continued for about six minutes when the reduction is complete. About fifty cubic centimeters of water are then added; also an excess of soda-lye and a few particles of zinc and the ammonia is distilled and collected in standard acid in the usual way.
The method of Ulsch can also be applied, according to Fricke, to the analysis of nitrates contained in drinking and drainage waters, and it is regarded by him as one of the best methods to be employed in such investigations.[317]
The method of Ulsch in this laboratory has given entirely satisfactory results, and is generally used in preference to other methods in cases where a considerable quantity of nitrates is present. It is based on the following reactions:
2KNO₃ + H₂SO₄ = K₂SO₄ + 2HNO₃ 2HNO₃ + 8H₂ = 2NH₃ + 6H₂O 2NH₃ + H₂SO₄ = (NH₄)₂SO₄.
REDUCTION BY THE ELECTRIC CURRENT.
=489. Method of Williams-Warington.=—From the losses which naturally occur during the evaporation of water, even with all the precautions noted, Warington was led to try some method for the determination of nitrates and nitrites in waters without previous concentration.[318] The reduction of these bodies by the copper-zinc couple formed the basis of these experiments, and they resulted in the following method of manipulation, which is based on a process devised by Williams.[319]
The method consists in boiling rapidly one liter of the rain water in a retort, with a little magnesia previously raised to a low red heat and then washed, until 250 cubic centimeters have distilled over. The residue is then made up to 800 cubic centimeters, transferred to a wide-mouthed, stoppered bottle supplied with strips of copper and zinc forming electric couples, and set aside, at a constant temperature of from 21°–24°, for three days. A measured portion of the solution is then distilled, and the ammonia determined in the distillate by nesslerizing.
This plan has two advantages: First, the ammonia, as well as the nitrogen as nitrates and nitrites, can be determined in the course of the same operation and in the same sample of water. For this purpose it is only necessary to fit the retort to an efficient condenser and to remove all ammonia from the apparatus by boiling distilled water in the retort before introducing the rain water. The distillate of 250 cubic centimeters from the rain water, as described above, is well mixed and the ammonia determined, in from twenty-five to one hundred cubic centimeters thereof, diluted to 150 cubic centimeters with ammonia-free water. Second, the nitrogen, as nitrates and nitrites, is determined directly and alone; the error of the determination is as small as nesslerizing admits of, since it is possible, if necessary, to distill 600 cubic centimeters of the boiled rain water corresponding to 750 cubic centimeters of the original, and thus obtain a full amount of ammonia for determination, even when the rain water has been poor in nitrates.
The determination of nitric nitrogen, in a given sample, by the above method gave a mean quantity of product of 0.162 part per million, while the determination, in the same lot of samples, by the modified Schloesing method gave 0.125 part per million. This result confirms the supposition that in the complete evaporation necessary to the manipulation of the Schloesing method there is a loss of nitrogen. The amount of nitrogen as nitrates and nitrites in the rain water at Rothamstead, for the twelve months ending April 1, 1888, was found, by the Schloesing method, to be 0.614 pound per acre, the total rain-fall being 21.96 inches. For the year ending April 1, 1889, by the copper-zinc method, it amounted to 0.917 pound per acre, the total rain-fall being 29.27 inches.
The amounts found in other localities are quite different from the above, as for instance, the mean of seven stations in Germany for thirteen years, beginning in 1864, showed 10.18 pounds of nitrogen per acre. The average amount for ten years at the observatory of Mont Sauris, near Paris, showed 12.36 pounds of nitrogen per acre. The average for three years at Lincoln, as determined by Professor G. Gray, shows one and six-tenths pounds of nitrogen per acre per annum. At Tokio, in Japan, Kellner found, for one year, 1.02 pounds per acre.
=490. Determination of the Ammonia.=—The method used at Rothamstead is to make one determination of ammonia in the whole of the distillate obtained, the strength of which is regulated by varying the amount introduced into the retort, so that it shall be equal to about two cubic centimeters of the standard ammonia solution. A 150 cubic centimeter cylinder is first filled with the rain water, and fifty cubic centimeters of nessler reagent added. The depth of tint indicates what quantity of rain water will be required for distillation. This having been determined, the appropriate volume of the rain water, provided it do not exceed 600 cubic centimeters, is placed in the retort described above, and the distillation continued until the 150 cubic centimeter cylinder is filled. The titration is made in the usual way.
=491. Preparation of the Copper-Zinc Couple.=—For 800 cubic centimeters of boiled rain water, prepared as described, six strips of zinc foil, four inches long by one and a quarter inches wide, are taken and bent at right angles along their center to obtain stiffness. The couple is cleansed and coated by washing in a series of five beakers containing, respectively, dilute solution of sodium hydroxid, very dilute sulfuric acid, a three per cent solution of copper sulfate, ordinary distilled water, and distilled water free from ammonia. Through these five beakers the zinc foil is successively passed. It is rinsed both after the alkali and the acid. But after the copper has been deposited, the strips are simply drained and carefully placed in the distilled water, it being difficult to rinse without removing the copper. The couples should be entirely submerged when placed in the rain water. The strips should remain in the copper sulfate solution long enough to be well covered with copper.
=492. Substitution of an Aluminum-Mercury Couple for Copper-Zinc.=—Ormandy and Cohen have proposed to use an aluminum-mercury couple for the copper-zinc in the process described above.[320]
This couple acts more quickly than the copper-zinc, and the results are equally as accurate. Nitrites are reduced in about one hour by this apparatus, while the zinc-copper couple of Gladstone and Tribe requires about six times as long. Aluminum foil, free of grease, should be used. The foil should be heated over a bunsen just before amalgamation. The clean, very thin foil is coated with mercury by shaking with a concentrated solution of mercuric chlorid. It should be prepared immediately before use.
The amalgamated foil is introduced into the sample of water to be analyzed, and left until all the aluminum is converted into oxid. The presence of the oxid favors the prevention of bumping during the subsequent distillation. The distilled ammonia, collected in dilute acid, is determined by nesslerizing, the free ammonia in the sample having been previously determined. The increase in ammonia is due to nitrates or nitrites reduced by the couple.
IODOMETRIC ESTIMATION OF NITRIC ACID IN NITRATES.
=493. Method of De Koninck and Nihoul.=—This process is applicable only in the absence of organic bodies and other reducing agents.
The principle on which it rests, as applied by McGowan, is as follows:[321]
When a fairly concentrated solution of a nitrate is warmed with an excess of pure, strong hydrochloric acid, the nitrate is completely decomposed, and the production of nitrosyl chlorid and chlorin is quantitative. The reaction, as shown by Tilden, is represented by the following equation:[322]
HNO₃ + 3HCl = NOCl + Cl₂ + 2H₂O.
One molecule of nitric acid thus yields two atoms of chlorin and one molecule of nitrosyl chlorid capable of setting free three atoms of iodin. The iodin can be estimated in the usual manner by titration with sodium thiosulfate. The nitrosyl chlorid is decomposed by the potassium iodid, nitric oxid escaping.
The apparatus employed is shown in Fig. 87.
A is a small, round-bottomed flask, into the neck of which a glass stopper, x, is accurately ground (with fine emery and oil). The capacity of the bulb is about forty-six cubic centimeters, and the length of the neck, from x to y, ninety millimeters. The first condenser is a simple tube, slightly enlarged at the foot into two small bulbs.[323] The length from a to b is 300 millimeters, from b to c 180 millimeters, and from e to f thirty millimeters. The capacity of the bulb B is twenty-five cubic centimeters, and the total capacity of the two bulbs and tube, up to the top of C, forty-one cubic centimeters. This condenser is immersed, up to the level of c, in a beaker full of water. D is a geissler bulb apparatus, E is a calcium chlorid tube, filled with broken glass, which acts as a tower and g is a small funnel, attached by rubber and clip to the branch =⟙= tube h. Between the =⟙= tube i and the wash-bottle for the carbon dioxid is placed a short piece of glass tubing, s, containing a strip of filter paper, slightly moistened with iodid of starch solution. This tube s is really hardly necessary, as no chlorin escapes backwards if a moderate current of carbon dioxid is kept passing, but it serves as a check. A glance at the joints o, p, and q, which are of narrow india-rubber tubing, is sufficient to show that, by using this arrangement, practically no rubber is exposed to the action of the chlorin. The tiny piece of rubber tubing at the joint o may be done away with, the narrower tube there being accurately ground into the wider one; this makes the condensing apparatus practically perfect.
The actual operation is performed in the following manner:
The evolution flask is washed and thoroughly dried, and the nitrate (say, about 0.25 gram of potassium nitrate) is tapped into it from the weighing tube. Two cubic centimeters of water are now added, and the bulb is gently warmed, so as to bring the nitrate into solution, after which the stopper of the flask is firmly inserted. About fifteen cubic centimeters of a solution of potassium iodid (one in four) are run into the first condensing tube, any iodid adhering to the upper portion of the tube being washed down with a little water, and five cubic centimeters of the same solution, mixed with eight to ten cubic centimeters of water, are sucked into the geissler bulbs whilst the glass in the tower E is also thoroughly moistened with the iodid. The geissler bulbs should be so arranged that gas only bubbles through the last of them, the liquid in the others remaining quiescent.
All the joints having been made tight the carbon dioxid is turned on briskly and passed through the apparatus until a small tubeful collected at l, over caustic potash solution, shows that no appreciable amount of air is left in it. The small outlet tube l, is now replaced by a calcium chlorid tube, filled with broken glass which has been moistened with the above-mentioned iodid solution, and closed by a cork through which an outlet tube passes, the object of this trap tube being to prevent any air getting back into the apparatus. The brisk current of carbon dioxid is continued for a minute or two longer, so as to practically expel all the air from this last tube. The stream of gas is now stopped for an instant, and about fifteen cubic centimeters of pure concentrated hydrochloric acid, free from chlorin, run into A through the funnel g (into the tube of which it is well to have run a few drops of water before beginning to expel the air from the apparatus), and A is shaken so as to mix its contents thoroughly. A slow current of carbon dioxid is now again turned on (one to two bubbles through the wash-bottle per second), and A is gently warmed over a burner. It is a distinct advantage that the reaction does not begin until the mixed solutions are warmed, when the liquid becomes orange-colored, the color again disappearing after the nitrosyl chlorid and chlorin have been expelled. The warming should be very gentle at first in order to make sure of the conversion of all the nitric acid, and also because the first escaping vapors are relatively very rich in chlorin; afterwards the liquid in A is briskly boiled. A very little practice enables the operator to judge as to the proper rate of warming. When the volume of liquid in A has been reduced to about seven cubic centimeters (by which time it is again colorless) the stream of carbon dioxid is slightly quickened and the apparatus allowed to cool a little. The burner is now set aside for a few minutes, and two cubic centimeters more of hydrochloric acid, previously warmed in a test-tube, run in gently through g; there is no fear either of the iodid solution running back, or of any bubbles of air escaping through y if this is done carefully. This is a precautionary measure, in case a trace of the liberated chlorin might have lodged in the comparatively cool liquid in the tube h. The carbon dioxid is once more turned on slowly and the liquid in A is boiled again until it is reduced to about five cubic centimeters. It is now only necessary to allow the apparatus to cool, passing carbon dioxid all the time, after which the contents of the condensers are transferred to a flask and titrated with thiosulfate. At the end of a properly conducted experiment, the glass in the upper part of tower E should be quite colorless and there should be only a mere trace of iodin showing in the lower part of the tower, while the liquid in the last bulb of the geissler apparatus ought to be pale yellow. During the operation, the stopper of A and the various joints can be tested from time to time by means of a piece of iodid of starch paper, and before disjointing it is well to test the escaping gas (say at m) in the same way, to make sure that all nitric oxid has been thoroughly expelled.
The method is capable of giving accurate results, but it can not be preferred to the reduction or colorimetric processes.
=494. Method of Gooch and Gruener.=—The principle on which this method rests depends on the decomposition of a nitrate in presence of a hot saturated solution of manganous chlorid and hydrochloric acid in an atmosphere of carbon dioxid.[324] The products of decomposition are passed into a solution of potassium iodid and the liberated iodin is titrated with standard sodium thiosulfate. The products of the reaction are chlorin, nitric oxid, and possibly nitrosyl chlorid, and under proper precautions the iodin set free is quantitively proportional to the weight of nitrate decomposed. The manganous mixture is acted on slowly at ordinary temperatures, but on heating, the nitrate is decomposed with the formation of a higher manganese chlorid and nitric oxid. When the heat is continued a sufficient length of time the chlorin from the higher chlorids is evolved and only manganous chlorid remains. During the heating the color of the solution passes from green to black and at the end the green color is restored. The apparatus employed is shown in Fig. 88.
A plain pipette bent as is shown in the figure serves as the generating flask and for the attachment on the one hand to the carbon dioxid apparatus and on the other to the system of absorption bulbs for containing the potassium iodid. The latter should be glass, sealed to the evolution bulb of the pipette to prevent the action of the evolved gases on organic materials. The point of the potassium iodid apparatus is drawn out so as to be pushed well into the second receiver, being held in place by a piece of rubber tubing. The third tube acts simply as a trap to exclude the air from the absorption apparatus. The first receiver contains in solution three grams, the second two, and the third one gram of potassium iodid. During the reaction the first receiver is kept cool by immersion in water. Before connecting the apparatus with the carbon dioxid generator the solution of manganous chlorid and afterwards the nitrate solution are drawn into the bulb of the pipette by gentle suction. After connecting the apparatus the current of carbon dioxid is started and kept up until all the air is expelled. Heat is then applied to the bulb of the pipette and the distillation continued until all the liquid has passed over. At the end of the reaction the contents of the receivers are united by disconnecting the apparatus from the carbon dioxid generator and passing water through the pipette. The introduction of the manganous chlorid into the mixture does not interfere with the titration of the iodin. This is accomplished in the usual way with sodium thiosulfate using starch as an indicator. The quantity of material used should contain about the amount of nitric acid that is found in two-tenths of a gram of potassium nitrate. This method, so similar to the preceding, is somewhat less complex, and, to that extent, preferable to it.
ESTIMATION OF NITRIC ACID BY COLORIMETRIC COMPARISON.
=495. Delicacy of the Method.=—The remarkable delicacy of those methods of chemical analysis, which depend on the production of a pronounced color, which can be compared with that produced by a known quantity of a given substance, has been long illustrated by the nesslerizing process for the estimation of ammonia. By such methods minute qualities of substances can be quantitively determined with great accuracy, when they would escape all effort for their estimation by gravimetric methods. Processes based on this principle are, therefore, peculiarly applicable to the detection and estimation of oxidized nitrogen in waters and soil extracts, whether they be present as nitric, nitrous, or ammoniacal compounds.
In the following paragraphs will be given with sufficient detail for the needs of the analyst, the principles and practice of the colorimetric comparison methods which have been approved as best by the experience of analysts. These methods are applicable especially to cases in which only minute quantities of the substances looked for are present, and where celerity of determination is especially desirable. They are, therefore, of especial value in the analysis of rain, drainage, and irrigation waters, and of soil extracts poor in oxidized nitrogen.
=496. Hooker’s Method.=—The quantitive action depends upon the deep green coloration given by nitric acid, when dissolved in sulfuric acid and carbazol.[325] Other oxidizing bodies, such as iron, chlorin, bromin, chromic acid, etc., give the same reaction, but not in such a prominent manner. Such bodies with the exception of chlorin and iron, are not often found in waters. In the application of the process, iron, if present in quantities greater than one-tenth part per one hundred thousand, must be removed. Chlorids also, even when present in very small quantities, interfere with the delicacy of the reaction and must be removed. Easily destructible organic matter tends to lower the result, but not materially, unless present in large excess. Calcium carbonate and sulfate, soda, and other alkalies, in the quantities in which they are usually present in water, do not affect the result. The following reagents are required:
1. Concentrated sulfuric acid.
2. An acetic acid solution of carbazol; diphenylimid, (C₆H₄\/C₆H₄/ NH.)
3. A sulfuric acid solution of carbazol.
4. Standard solutions of potassium nitrate.
5. A solution of aluminum sulfate.
6. A solution of silver sulfate.
1. The sulfuric acid, used for all purposes in the process, should be entirely free from nitrogen oxids. It may be readily tested by dissolving in it a small quantity of carbazol. If the solution be at first golden-yellow or brown, the acid is sufficiently pure; if it be green or greenish, another and better sample must be taken. It is essential also that the specific gravity of the acid be fully 1.84, and it is well to ascertain that this is really the case.
2. The acetic acid solution of carbazol is prepared by dissolving six-tenths gram in about ninety cubic centimeters of strongest acetic acid, by the aid of gentle heat. It is allowed to cool, and is then made up to 100 cubic centimeters by the further addition of acetic acid. The exact strength of this solution, is of no material importance to the success of the process, and the above proportions have been selected principally because they are convenient. The solution will remain unchanged for several months. The use of this solution merely facilitates the preparation of that next described, which will not keep, and has, consequently, to be freshly prepared for each series of determinations.
3. The sulfuric acid solution of carbazol is easily made in a few seconds, but it is advisable to allow it to stand from one and one-half to two hours before using. It is prepared by rapidly adding fifteen cubic centimeters of sulfuric acid, to one cubic centimeter of the above-described acetic acid solution. This quantity usually suffices for from two to three nitrate estimations. When freshly prepared it is golden-yellow or brown; it changes gradually, however, and in the course of one and one-half or two hours it becomes olive-green. This change is probably due to traces of oxidizing agents, which occur in the sulfuric and acetic acids, and which, although not present in sufficient quantity to act immediately, gradually bring about the reaction described. The greenish color does not interfere with the process, as might at first be supposed; on the contrary, the solution is not sensitive to small quantities of nitric acid until it has undergone the change to olive-green, and it is for this reason, that it should be prepared about two hours before required for use. This solution may be thoroughly depended on for six hours after preparation. The intensities of color produced by the more concentrated solutions of nitrates after this time, gradually approach each other and become ultimately the same.
4. The standard solutions of potassium nitrate are very readily prepared. The solutions which are to be compared directly with the waters examined, may be prepared as required, but if many determinations are to be made with a variety of waters, it will be found best to prepare a complete series, differing from each other by 0.02 part nitrogen in 100,000. This series may include solutions containing quantities of nitrogen in 100,000 parts, represented by all the odd numbers from 0.03 up to 0.39. It will be found convenient to prepare them in quantities of 100 cubic centimeters at a time, from a stock solution of potassium nitrate which contains 0.00001 gram nitrogen, or 0.000045 nitric acid in one cubic centimeter. Each cubic centimeter of this solution, when diluted to 100 cubic centimeters, represents 0.01 nitrogen in 100,000, and consequently if it is desired to make a solution containing 0.35 part nitrogen in 100,000, thirty-five cubic centimeters are taken and made up to 100 cubic centimeters, and so on. The solution of potassium nitrate (b) is best prepared from a stronger one (a) containing 0.0001 gram nitrogen to the cubic centimeter, or 0.7214 gram potassium nitrate to the liter; 100 cubic centimeters of (a) made up to one liter give the solution (b). It is obvious that the series of solutions, above described, could be made directly from (a), but by first making (b), greater accuracy is secured.
5. For purposes which will be presently described, a solution of aluminum sulfate is required, containing five grams to the liter. The salt used must be free from chlorin and iron; and the solution should give no reaction when tested with carbazol.
6. The solution of silver sulfate is required for the removal of chlorin from the water or soil extract to be examined. It is prepared by dissolving 4.3943 grams of the salt in pure distilled water and making up to one liter. The sulfate is preferably obtained by dissolving metallic silver in pure sulfuric acid. The solution should be tested with carbazol in the same way as will be presently described for water; if perfectly pure, no reaction will be obtained. As silver sulfate is often prepared by precipitation from the nitrate, it is very apt to contain nitric acid, and consequently, if the source of the salt be unknown, this test should on no account be omitted.
The analytical process is carried on as follows:
Two cubic centimeters of the water are carefully delivered by means of a pipette into the bottom of a test-tube; four cubic centimeters of sulfuric acid are added, and the solution thoroughly mixed by the help of a glass rod. The test-tube is then immersed in cold water, and when well cooled, one cubic centimeter of the sulfuric acid solution of carbazol is added, and the whole again mixed as before. The intensity of the color is now observed, and a little experience enables a fairly good opinion to be formed of the quantity of nitric acid present. Suppose that the water be roughly estimated to contain about 0.15 part nitrogen per 100,000; in such a case solutions of potassium nitrate containing 0.11, 0.15, 0.19 part nitrogen are selected from the series. Two cubic centimeters are taken from each, and treated, side by side, with a fresh quantity of the water, precisely as described for the preliminary experiment, the various operations being performed as nearly simultaneously as possible with each of the samples, and under precisely similar conditions. Two or three minutes after the carbazol has been added, the intensity of the color of each is observed. If that given by the water is matched by any of the standard solutions, the estimation is at an end. Similarly, if it falls between two of these, the mean may be taken as representing the nitrogen present in cases in which great accuracy is not required. If this be done, the maximum error will be 0.02 part nitrogen, or 0.09 part nitric acid per 100,000. If greater exactness be required, or it be found that the color given by the water is either darker or lighter than that given by all the standard solutions, a new trial must be made. In such a case the water must be again tested simultaneously with the solutions with which it is to be compared. This is rendered necessary principally for the reason that the shade of the solutions to which the carbazol has been added is apt to change on standing. Hence it is desirable that the water, and the standard potassium nitrate with which it is to be compared, should have the carbazol added at as nearly the same time as possible. When finally the color falls between that given by any two consecutive members of the standard potassium nitrate series, the estimation may be considered at an end, and the mean of these solutions taken as representing the nitrogen present.
The greatest neatness should be observed in all steps of the analysis. The quantity of water operated upon is so small that if the greatest care be not exercised throughout, sources of error may be readily introduced. The test-tubes should be rinsed out with nitrate-free water before being used and then dried. The tint should be determined by looking through the tube and not through the length of the column of liquid.
Influence of Nitrites.—If the quantity of nitrous acid in the water is known a correction can be applied for nitrates by deducting one-fifth of the number found for nitrites when estimated as nitrates.
Influence of Iron.—Although ferrous salts give no reaction with carbazol, nitrates are apt to be overestimated in their presence. Oh the other hand, ferric compounds, like other oxidizing agents, may give a characteristic green color with carbazol. In all cases when iron is present in any considerable quantity it is best to remove it by rendering the water slightly alkaline, evaporating to dryness, and redissolving the soluble residue until the solution reaches the original volume.
Influence of Chlorids.—The presence of chlorids furnishes by far the most serious source of error in the process by intensifying the action of the nitric acid. If, however, nitrates be absent chlorids give no reaction with carbazol. The chlorids are removed by a standard silver sulfate solution, the quantity of chlorids present having been first determined by a standard silver nitrate solution. For this purpose an ordinary sugar flask can be employed marked at 100 and 110 cubic centimeters. This flask is filled to the 100 cubic centimeter mark with the water to be examined; the necessary quantity of silver sulfate is added and then two cubic centimeters of the solution of aluminum sulfate, previously described, and the contents of the flask brought up to 110 cubic centimeters by the addition of pure distilled water. The whole is shaken up and filtered, the first portion of the filtrate being rejected. The aluminum sulfate by reacting with the carbonates usually present in the water and producing the precipitation of alumina, facilitates the removal of the precipitated silver chlorid.
The above-described method on account of its delicacy is not well suited to aqueous solutions of soils except where the quantity of nitric nitrogen present is extremely minute.
Hooker also first suggested the use of diphenylamin for detecting the presence of nitrates,[326] a method afterwards worked out by Spiegel.[327]
In the variation of the method as practiced by Rideal the standard potassium nitrate and the pure sulfuric acid mentioned below are required, and in addition, the following reagents:[328]
(a) Silver sulfate solution containing 4.3945 grams per liter.
(b) Aluminum sulfate solution free from chlorids and iron, five grams per liter.
(c) Carbazol solution; six-tenths gram carbazol dissolved in glacial acetic acid and made up to 100 cubic centimeters with the glacial acid. For use, one cubic centimeter of this solution is withdrawn by a pipette and mixed with fifteen cubic centimeters of pure redistilled sulfuric acid.
The process is carried out as follows: To 100 cubic centimeters of water the amount of chlorin which has been previously ascertained is removed by the silver sulfate solution. Two cubic centimeters of the aluminum sulfate are added and the whole made up to a convenient volume, say about 110 cubic centimeters. The liquid is filtered and two cubic centimeters of the filtrate taken for an estimation of nitrates. To the two cubic centimeters are added four cubic centimeters of concentrated sulfuric acid and the mixture cooled.
One cubic centimeter of the carbazol solution in sulfuric acid is added and a bright green color appears in a few moments, if nitrates are present. Comparison is made with solutions of standard potassium nitrate.
=497. Phenylsulfuric Acid Method.=—Rideal also proposes a variation of the method described by Hooker, which consists in the substitution of phenylsulfuric acid for carbazol.[329]
The solutions required are:
(a) A standard solution of potassium nitrate containing 0.7215 gram of the pure crystallized salt in a liter of water.
(b) Phenylsulfuric acid, (acid phenyl sulfate,) prepared by dissolving fifteen grams of pure crystallized phenol in 92.5 cubic centimeters of pure, redistilled sulfuric acid free from nitrates and diluted with seven and one-half cubic centimeters of water.
The process is conducted as follows:
A known volume of water, from twenty-five to one hundred cubic centimeters, according to its richness in nitrates, is evaporated to dryness in a porcelain dish, one cubic centimeter of phenylsulfuric acid added then one cubic centimeter of pure water and three drops of strong sulfuric acid and the mixture gently warmed. A yellow color shows the presence of nitrates. Dilute to about twenty-five cubic centimeters with water and add ammonia in slight excess. Pour into a narrow nessler tube and add the washings and make up to 100 cubic centimeters. Imitate the color of the solution with the standard potassium nitrate treated with the same reagents.
The phenylsulfuric acid should be prepared some time before use, as the fresh solution imparts a greenish tint to the yellow of the ammonium picrate formed.
=498. Variation of Leffmann and Beam.=—The phenyl sulfate process, as described by Leffmann and Beam, is conducted as follows:[330]
Solutions Required.—Acid phenyl sulfate: 18.5 cubic centimeters of strong sulfuric acid are added to one and one-half cubic centimeters of water and three grams of pure phenol. Preserve in a tightly-stoppered bottle.
Standard potassium nitrate: 0.722 gram of potassium nitrate, previously heated to a temperature just sufficient to fuse it, is dissolved in water, and the solution made up to 1000 cubic centimeters. One cubic centimeter of this solution will contain 0.0001 gram of nitrogen.
Analytical Process.—A measured volume of the water is evaporated just to dryness in a platinum or porcelain basin. One cubic centimeter of the acid phenyl sulfate is added and thoroughly mixed with the residue by means of a glass rod. One cubic centimeter of water, and three drops of strong sulfuric acid are added, and the dish gently warmed. The liquid is then diluted with about twenty-five cubic centimeters of water, ammonium hydroxid added in excess, and the solution made up to 100 cubic centimeters.
The reactions are:
Acid phenyl sulfate. Trinitrophenol (picric acid). HC₆H₅SO₄ + 3HNO₃ = HC₆H₂(NO₂)₃O + H₂SO₄ + 2H₂O.
Ammonium picrate. HC₆H₂(NO₂)₃O + NH₄HO = NH₄C₆H₂(NO₂)₃O + H₂O.
The ammonium picrate imparts to the solution a yellow color, the intensity of which is proportional to the amount present.
Five cubic centimeters of the standard solution of potassium nitrate are similarly evaporated in a platinum dish, treated as above, and made up to 100 cubic centimeters. The color produced is compared to that given by the water, and one or the other of the solutions diluted until the tints of the two agree. The comparative volumes of the liquids furnish the necessary data for determining the amount of nitrate present, as the following example will show:
Five cubic centimeters of standard nitrate are treated as above, and made up to 100 cubic centimeters, representing 0.0005 gram nitrogen.
Suppose 100 cubic centimeters of water similarly treated are found to require dilution to 150 cubic centimeters before the tint will match that of the standard; then
100 : 150 :: 0.005 : 0.0075
i. e., the water contains seven and one-half milligrams of nitrogen as nitrate per liter.
The ammonium picrate solution keeps very well, especially in the dark. A good plan, therefore, is to make up a standard solution equivalent to, say, ten milligrams of nitrogen as nitrate per liter, to which the color obtained from the water may be directly compared.
The results obtained by this method are quite accurate. Care should be taken that the same quantity of acid phenyl sulfate be used for the water and for the comparison liquid, otherwise different tints instead of depths of tints are produced.
With subsoil and other waters probably containing much nitrates, ten cubic centimeters of the sample will be sufficient for the test, but with river and spring waters, twenty-five to one hundred cubic centimeters may be used. When the organic matter is sufficient to color the residue, it will be well to purify the water by addition of alum and subsequent filtration, before evaporating. The method may also be used to determine small quantities of nitrates in aqueous extracts of soils when the quantity is too small for estimation by the ferrous chlorid or reduction processes.
=499. Variation of Johnson.=—The ammonium picrate method has given very satisfactory results as practiced by Johnson, who varies the process as described below.[331]
The standard solution of potassium nitrate is prepared by dissolving 0.7215 gram of the pure salt in a liter of distilled water. Dilute 100 cubic centimeters of this solution to one liter with distilled water. Ten cubic centimeters of this dilute solution contain nitrogen equivalent to one part as nitrates in 100,000.
The Solution of Acid Phenyl Sulfate.—This is prepared by pouring two parts by measure of pure crystallized phenol liquefied by hot water into five parts by measure of pure concentrated sulfuric acid and digesting the whole in the water-bath for eight hours. After cooling, add one and one-half volumes of distilled water and one-half volume strong hydrochloric acid to each volume of the above mixture.
The analytical processes are carried on as follows: Ten cubic centimeters of the water under examination and ten cubic centimeters of the standard potassium nitrate are placed in small beakers and put near the edge of a hot plate. When nearly evaporated they are put on the top of the water-bath and left there until completely dry. The residue, in each case, is then treated with one cubic centimeter of the acid phenyl sulfate and the beakers placed on the top of the water-bath. In good water, a red color ought not to appear for about ten minutes.
After standing about fifteen minutes, the beakers are removed, the contents of each washed successively into 100 cubic centimeter flasks, about twenty cubic centimeters of 0.96 per cent. ammonia added, and the 100 cubic centimeters made up by the addition of water and the yellow liquid transferred to the nessler glass and the tints appropriately compared.
=500. Estimation of Nitric in Presence of Nitrous Acid.=—The detection of nitrous in presence of nitric acid can be accomplished by the method proposed by Griess, as described further on, through the development of azocolors, with metaphenylenediamin and other bodies, which are not produced under similar conditions by nitric acid. The detection and estimation of nitric in the presence of nitrous acid, however, is not so easy. Lunge and Lwoff propose brucin for this purpose, which, contrary to most authorities, does not give the red-yellow color with nitrous acid.[332] The reagent is prepared by dissolving two-tenths gram of brucin in 100 cubic centimeters of sulfuric acid, pure and concentrated. It is almost impossible to prepare a sulfuric acid which does not give a trace of color with brucin; but with the purest acids this trace may be neglected.
A solution of nitrate is also prepared containing 0.01 milligram of nitrogen as nitric acid in one cubic centimeter. It is made by dissolving 0.0721 gram of pure potassium nitrate in 100 cubic centimeters of distilled water, and diluting ten cubic centimeters thereof with pure concentrated sulfuric acid to 100 cubic centimeters. Both solutions are conveniently preserved in burettes with glass stop-cocks. The liquid to be tested for nitric acid should be mixed with sulfuric acid in such a way that the mixture will have a specific gravity of one and seven-tenths. If the liquid to be tested is water, this concentration is reached by adding three times its volume of the strong acid. For the comparison of colors, cylinders of colorless glass are employed, marked at fifty cubic centimeters. They should be about twenty-four centimeters high and extend about ten centimeters above the mark. There is placed in the cylinder one cubic centimeter of the solution of nitrate in sulfuric acid, and the same quantity of the brucin mixture, and it is filled to the mark with pure sulfuric acid. The contents of the cylinder are poured into a flask and warmed at from 70°–80°, until the final yellow tint is secured, and then poured into the cylinder again. The liquid to be tested is treated in exactly the same way. The tints are then equalized by pouring out a part of the contents of the deeper colored cylinder, taking account of the volume, and filling up with pure concentrated sulfuric acid.
In this manner the content of nitric acid in the liquid under examination can be compared directly with the solution of potassium nitrate of known strength. The coloration is distinctly produced with 0.01 milligram in fifty cubic centimeters of liquid, at least three-fourths of which must be sulfuric acid.
=501. Piccini Process.=—The method proposed by Piccini may also be used.[333]
About five cubic centimeters of the nitrite solution are placed in a small beaker, some pure urea dissolved therein and a few drops of sulfuric acid, and then held in boiling water for three minutes. The nitrous acid is thus completely destroyed. Ammonium chlorid may be substituted for urea. The reaction is given on page 478. The nitric acid present is then determined by diphenylamin or other suitable reagents. Diphenylamin reacts both with nitrous and nitric acids, producing a violet tint. Warington calls attention to a slight difference, however, in its deportment with these two acids. When the solution of the reagent is not too strong a drop of it produces but little turbidity when added to water or to a solution containing nitric acid. When, however, nitrous acid is present, a cream-colored turbidity is produced. The violet color also appears at once on adding sulfuric acid when a nitrite is present, while in the case of nitrates, more sulfuric acid is required, except when the solution is very strong. In this connection, it must not be forgotten that in heating nitrites with urea or ammonium chlorid in the presence of a slight excess of sulfuric acid a trace of nitric acid may be formed.
ESTIMATION OF NITROUS ACID BY COLORIMETRIC COMPARISON.
=502. Application of the Method.=—The most minute traces of nitrous acid may be detected by colorimetric methods and the determination of the quantity present may be approximated with great exactness by comparison with a solution of a nitrite of known strength. Especially in following the progress of nitrification is this method, in some of its forms, of essential importance. In delicacy and celerity it has the same advantages as the colorimetric methods for the determination of nitric acid.
=503. Metaphenylenediamin Method.=—This process depends upon the development of a yellow color in water containing nitrous acid on the addition of a reagent containing metaphenylenediamin; m-C₆H₄(NH₂)₂. This variety of the phenylenediamins is readily obtained from common dinitrobenzene. It melts at 63° and boils at 287°. In order to preserve the reagent in shape for use it should be prepared in the following manner:
Dissolve two grams of the chlorid in ten cubic centimeters of ammonia, and place the solution in a glass-stoppered flask. To this solution are added five grams of powdered animal-black, and the whole vigorously shaken. After allowing to settle, the shaking is repeated at intervals of an hour, three or four times, and the flask then allowed to remain at rest for twenty-four hours.
The supernatant liquid is generally sufficiently decolorized by this treatment. If not, the shaking and subsidence must be repeated until a completely colorless liquid is obtained. The solution can be kept, indefinitely, in contact with the animal-black. Aqueous and alcoholic solutions of the salt can not be kept.
The test is applied by mixing five drops of the reagent with five cubic centimeters of sulfuric acid. The mixture must be colorless. To the mixture add 100 cubic centimeters of the water to be tested, and heat on the water-bath for five minutes. A yellow coloration indicates the presence of nitrous acid.
The metaphenylenediamin test is fairly satisfactory in perfectly colorless waters and aqueous extracts. Many waters and soil extracts, however, have a yellowish tint, and this interferes in a marked way with a proper judgment of the yellow triaminazobenzol developed in the application of the above test.
The decoloration of such waters by means of sodium carbonate or hydroxid and alum, is a matter of some difficulty and not wholly without action on the nitrites which may be present. The method, therefore, is inferior to the one next described.
=504. Sulfanilic Acid and Naphthylamin Test for Nitrous Acid.=—A very delicate test for the presence of nitrous acid, first described by Griess, is the coloration produced thereby in an acid solution of sulfanilic acid and naphthylamin.[334]
Sulfuric or acetic acid may be used as the acidifying agent, preferably the latter. The solutions are prepared as follows:
(1) Dissolve one-half gram of sulfanilic acid in 150 cubic centimeters of dilute acetic acid.
(2) Boil one-tenth gram of naphthylamin with twenty cubic centimeters of water, decant the colorless solution from the residue and acidify it with 150 cubic centimeters of dilute acetic acid.
The two solutions may at once be mixed and preserved in a well-stoppered flask. The action of light on the mixture is not hurtful, but air should be carefully excluded because of the traces of nitrous acid which it may contain. Whenever the mixed solutions show a red tint it is an indication that they have absorbed some nitrous acid. The red color may be discharged and the solution again fitted for use by the introduction of a little zinc dust, and shaking.
The water, or aqueous solution of a soil, to be tested for nitrites, in portions of about twenty cubic centimeters, is treated with a few cubic centimeters of the mixed reagent and warmed to 70°–80°. If nitrous acid, in the proportion of one part to one million be present, the red color will appear in a few minutes. If the content of nitrous acid be greater, e. g., one part in one thousand, only a yellow color will be produced, unless a greater quantity of the reagent be used.
Leffmann and Beam recommend the following method of conducting the determinations.[335]
Solutions required:
Naphthylammonium Chlorid.—Saturated solution in water free from nitrites. It should be colorless; a small quantity of animal charcoal allowed to remain in the bottle will keep it in this condition.
Paraamidobenzene Sulfonic Acid (Sulfanilic Acid).—Saturated solution in water, free from nitrites.
Hydrochloric Acid.—Twenty-five cubic centimeters of concentrated pure hydrochloric acid added to seventy-five cubic centimeters of water, free from nitrites.
Standard Sodium Nitrite.—0.275 gram pure silver nitrite is dissolved in pure water, and a dilute solution of pure sodium chlorid added until the precipitate ceases to form. It is then diluted with pure water to 250 cubic centimeters and allowed to stand until clear. For use, ten cubic centimeters of this solution are diluted to 100. It is to be kept in the dark.
One cubic centimeter of the dilute solution is equivalent to 0.00001 gram of nitrogen.
The silver nitrite is prepared in the following manner: A hot concentrated solution of silver nitrate is added to a concentrated solution of the purest sodium or potassium nitrite available, filtered while hot and allowed to cool. The silver nitrite will separate in fine needle-like crystals, which are freed from the mother-liquor by filtration with the aid of a filter pump. The crystals are dissolved in the smallest possible quantity of hot water, allowed to cool and crystallize, and again separated by means of the pump. They are then thoroughly dried in the water-bath, and preserved in a tightly-stoppered bottle away from the light. Their purity may be tested by heating a weighed quantity to redness in a tared, porcelain crucible and noting the weight of the metallic silver. One hundred and fifty-four parts of silver nitrite leave a residue of 108 parts of silver.
Analytical Process.—One hundred cubic centimeters of the water are placed in one of the color-comparison cylinders, the measuring vessel and cylinder having previously been rinsed with the water to be tested. By means of a pipette, one cubic centimeter each of the solutions of sulfanilic acid, dilute hydrochloric acid, and naphthylammonium chlorid is dropped into the water in the order named. It is convenient to have three pipettes for this test, and to use them for no other purpose. In any case the pipette must be rinsed out thoroughly with nitrite-free water each time before using, as nitrites, in quantity sufficient to give a distinct reaction, may be taken up from the air.
One cubic centimeter of the standard nitrite solution is placed in another clean cylinder, made up with nitrite-free water to 100 cubic centimeters and treated with the reagents, as above.
In the presence of nitrites a pink color is produced, which, in dilute solutions, may require half an hour for complete development. At the end of this time the two solutions are compared, the colors equalized by diluting the darker, and the calculation made as explained under the estimation of nitrates.
The following are the reactions:
Paraamidobenzene Nitrous acid. Paradiazobenzene sulfonic acid. sulfonic acid. C₆H₄NH₂HSO₃ + HNO₂ = C₆H₄N₂SO₃ + 2H₂O.
Naphthylammonium Azoalphaamidonaphthalene parazobenzene chlorid. sulfonic acid. C₆H₄N₂SO₃ + C₁₀H₇NH₃Cl = C₁₀H₆(NH₂)NNC₆H₄HSO₃ + HCl.
The last named body gives the color to the liquid.
The method pursued by Tanner, in the preparation of the reagent, is as follows:
Sulfanilic acid is prepared by mixing thirty grams of anilin slowly, with sixty grams of fuming sulfuric acid, in a porcelain dish. The brown, sirupy liquid formed is carefully heated until quite dark in color, and until the evolution of sulfurous fumes is noticed. After cooling, the thick, semi-fluid mass is poured into half a liter of cold water and allowed to stand for some hours. The liquid portion is then decanted from the nearly black undissolved crystalline mass. To the residue half a liter of hot water is added and allowed to stand until cold, and the liquid again decanted. The undissolved portion is then treated with one liter of hot water and filtered. The filtrate is treated with animal charcoal to decolorize it, and allowed to stand for twenty-four hours and again filtered, the filtrate diluted to 1,500 cubic centimeters and used as required. This solution tends to turn pink on keeping, and thus its color interferes with the delicacy of the test, and a small amount of animal-char is kept in a small bottle containing the portion for immediate use, and this bottle is filled, from time to time, from the larger one.
The solution of naphthylamin hydrochlorate is made with one gram of the salt dissolved in 100 cubic centimeters of water. The solution is to be occasionally filtered, and not more than 100 cubic centimeters should be prepared at a time.
The analytical operations are carried on as follows:
A standard solution of pure potassium nitrite, made from the silver salt in distilled water perfectly free from nitrites, is placed in a color-glass, similar to those used in the nessler reaction, together with a second glass containing the water to be tested. These glasses should be marked to hold 100 cubic centimeters at the same depth. To each of the tubes a few drops of pure hydrochloric acid are added and two cubic centimeters of the sulfanilic solution. Afterwards, to each tube are added two cubic centimeters of the solution of naphthylamin hydrochlorate, and it is allowed to stand for twenty minutes, at the end of which time the color should be fully developed. Each tube is covered by a piece of glass in order to prevent access of air. It is unnecessary to add that the standard solutions of nitrite of different strength should be employed until the one is found which resembles, as nearly as possible, the color developed in the sample of water under examination.
=505. Lunge and Lwoff’s Process for Nitrous Acid.=—The reaction of nitrous acid with α naphthylamin, first described by Griess, may be made reliable, quantitatively, by proceeding as below:[336]
Boil 0.100 gram of pure white α naphthylamin for fifteen minutes with 100 cubic centimeters of water, add five cubic centimeters of glacial acetic acid, or its equivalent of dilute acid, and afterwards one gram of sulfanilic acid dissolved in 100 cubic centimeters of hot water. The mixture is kept in a well-closed flask. A slight red tint in the mixture is of no significance, inasmuch as this completely disappears when one part of it is mixed with fifty parts of the liquid to be examined. If the coloration be very strong it can be removed by adding a little zinc dust. One cubic centimeter of this reagent will give a distinct coloration with 0.001 milligram of nitrous nitrogen in 100 cubic centimeters of water.
The analysis is conducted in cylinders of white glass marked at fifty cubic centimeters. One cubic centimeter of the above reagent is placed in each of two cylinders with forty cubic centimeters of water and five grams of solid sodium acetate. In one of the cylinders is placed one cubic centimeter of a normal solution of a nitrite prepared by dissolving 0.0493 gram of pure sodium nitrite corresponding to ten milligrams of nitrogen in 100 cubic centimeters of water, and adding ten cubic centimeters of this solution to ninety cubic centimeters of pure sulfuric acid. This secures a normal solution of nitrosylsulfuric acid, of which each cubic centimeter corresponds to 0.01 milligram of nitrogen.
In the other cylinder is placed one cubic centimeter of the solution to be examined, and the contents of both cylinders are well mixed so that the nitrous acid in a nascent state may act on the reagent. The colors are compared after any convenient period, but, as a rule, after five minutes.
The chief improvement made by Lunge and Lwoff on the method of Griess is in keeping the reagent in a mixed state ready for use, by means of which any nitrous impurities in the components thereof are surely indicated. Its advantage over the method of Ilosvay[337] consists in using the comparative normal nitrite solution as nitrosylsulfuric acid, in which state it is much more stable.
=506. Estimation of Nitrous Acid with Starch as Indicator.=—The method of procedure, depending on the blue color produced in a solution of starch in presence of a nitrite and zinc iodid when treated with sulfuric acid, is not of wide application on account of the interference produced by organic matter. The soil extract or water is treated in a test-tube, with a few drops of starch solution and some zinc iodid, to which is added some sulfuric acid. The decomposition of the nitrite is attended with the setting free of an equivalent amount of iodin which gives a blue coloration to the starch solution. The depth of the tint is imitated by treating a standard solution of nitrite in a similar way until the proper quantity is found, which gives at once the proportion of nitrite in the sample examined. This process, however, is scarcely more than a qualitative one.
=507. Estimation of Nitrites by the Method of Chabrier.=—In order to make the estimation of the evolved nitrous acid more definite by the iodin method, Chabrier has elaborated a plan for titrating it with a reducing agent.[338]
The substance chosen for this purpose is sodium hyposulfite. In point of fact, it is not the nitrous acid which is attacked by the hyposulfite, but the equivalent amount of free iodin representing it. In the case of a soil where the quantity of nitrites is usually very small, it is well to take as much as one kilogram. The extraction should be made rapidly, with water, free of nitrites, in order to avoid any reducing action on the nitrates which may be present. In the case of water, from five to ten liters should be evaporated to a small volume. The concentration should take place in a large flask, rather than in an open dish, in order to avoid any possibility of the absorption of nitrites produced by combustion. When the volume has been reduced to about 100 cubic centimeters it is transferred to a small flask and the concentration continued until only ten or fifteen cubic centimeters are left. The residue is filtered into a woulff bottle, F, Fig. 89, of about 100 cubic centimeters capacity.
One of the side tubulures carries a burette, B, containing five per cent sulfuric acid, the other one filled with a hyposulfite solution of known strength. The middle tubule serves to introduce a glass tube through which carbon dioxid or illuminating gas passes for the purpose of driving out the air from the solution and the flask. If carbon dioxid be used it should be generated by the action of sulfuric acid on marble. The cork holding this is furnished with a slot or valve to permit the exit of the air and the excess of the gas.
Before inserting the middle stopper, a few cubic centimeters of potassium iodid solution and a few drops of thin starch paste are added, the potassium salt being always used in excess of the nitrite supposed to be present.
After the air has all been expelled from the flask the analytical process is commenced, the carbon dioxid current being slowly continued. At first, a few drops of the dilute sulfuric acid are allowed to flow into the flask. As soon as the liquid is colored blue a sufficient quantity of the thiosulfate solution is added to discharge the color. The successive addition of acid and thiosulfate is continued until another portion of the acid fails to develop the blue color, thus indicating that all the nitrite has been decomposed. From the volume of thiosulfate used the quantity of nitrite is calculated.
The Thiosulfate Solution.—The thiosulfate solution is conveniently prepared, when a large number of analyses is to be made, by dissolving twenty-five grams of pure crystallized sodium thiosulfate in 100 cubic centimeters of water and diluting any convenient part thereof to 100 or 1,000 cubic centimeters, according to the supposed strength of nitrite solution under examination.
For fixing the strength of the solution dissolve 3 348 grams of pure iodin in a solution of potassium iodid and make the volume up to one liter. Each cubic centimeter of this solution corresponds to one milligram of nitrous acid. A given volume of the iodin solution is titrated against the thiosulfate, but it is best not to add the starch paste until the greater part of the iodin has been removed. The starch paste is then added and the titration continued until the blue color has been discharged. Ten cubic centimeters of the iodin solution is a convenient quantity for the titration and the thiosulfate should be diluted by adding to ten cubic centimeters of the solution mentioned above, 990 cubic centimeters of water. Each liter of this dilute solution contains two and a half grams of the sodium thiosulphate.
Example.—Let us suppose that it has required 21.3 cubic centimeters of thiosulfate to absorb ten cubic centimeters of the iodin solution; further that ten liters of water have been evaporated and titrated as described above, and that the volume of thiosulfate employed was 13.8 cubic centimeters. From this is derived the following formula: (13.8 × 10)/(21.3) = 6.48 milligrams of nitrous acid; or 0.648 milligram per liter.
=508. Estimation of Nitrous Acid By Coloration of Solution of Ferrous Salt.=—This method, due to Picini is based on the production of the well-known brown color formed by the action of nitric oxid on a ferrous salt.[339] The nitrite is decomposed by heating with acetic acid while nitrates thus treated do not develop the reaction. The tint produced is imitated as above by testing against a standard solution of nitrite. Ferrous chlorid is to be preferred to other ferrous salts for the above purpose. The process should be carried on in solutions free of air.
=509. Estimation of Nitrous Acid By Decomposition with Potassium Ferrocyanid.=—The method of Schaeffer was first described in 1851, but little attention has been paid to it since. The method has lately been brought into notice again by Deventer.[340]
The reaction depends upon the decomposition of nitrous acid by potassium ferrocyanid in the presence of acetic acid with the formation of potassium ferricyanid and acetate, and nitric oxid. The reaction is expressed by the following equation:
2K₄FeCy₆ + 2HNO₂ + 2C₂H₄O₂ = K₆Fe₂Cy₁₂ + 2KC₂H₃O₂ + 2NO + 2H₂O.
A eudiometer with a glass stop-cock is arranged as shown in Fig. 90. The lower part of the eudiometer is closed with a rubber stopper carrying a glass tube which ends in the pan f as shown at e. The eudiometer is filled to the stop-cock with a solution of potassium ferrocyanid of about fourteen per cent strength. The dish f is also filled up to the height indicated in the figure with the same solution. The solution of nitrite is used in such quantities that the nitric oxid evolved will occupy a space of about twenty cubic centimeters. The whole eudiometer should contain about fifty-seven cubic centimeters. The nitrite solution is added to the eudiometer by means of a funnel, a. The vessel containing it is washed out with a little water and then with acetic acid and finally with a few cubic centimeters of strong potassium ferrocyanid solution. The last fluid flows through the solution of nitrite and acetic acid and thus mixes it with the solution already in the eudiometer. The liquids reacting on each other float together on the strong ferrocyanid solution and each one of them is at once pressed downward by the gases which are evolved. When the evolution of gas becomes slower the apparatus should be shaken for about twenty minutes, moving it back and forth without taking the bottom of it out of the dish. When there is no longer any evolution of gas, water is added through a slowly, until the heavy potassium ferrocyanid solution is almost completely driven out of the eudiometer. The opening of the tube at e is then closed with the thumb, the apparatus is taken out of the dish, shaken for some time in a vertical direction and again placed in the dish. Water of any required temperature is now allowed to flow through the jacket, g, h, until the temperature is constant, when the volume of nitric oxid is read. The whole experiment can be performed in less than an hour. Operating in this way, at the end there is in the eudiometer a liquid which is not very different from water and one whose coefficient of solubility for nitric oxid is practically the same as that of water. The gas volume read is to be corrected for temperature, pressure, tension of the aqueous vapor, height of the water column in the eudiometer, and, after the end of the calculation, five per cent of the volume of water remaining in the eudiometer is to be added to the volume of gas obtained. This is to compensate for the volume of the gas absorbed by the water. The method gives good quantitive results.
=510. Method of Collecting Samples of Rain Water for Analysis.=—Warington collects rain water in a large leaden gauge having an area of 0.001 of an acre.[341] Of the daily collection of rain, dew, and snow water, an aliquot part amounting to a gallon for each inch of precipitation is placed in a carboy; at the end of each month the contents of the carboy are mixed, and a sample taken for analysis. In the carboy receiving the rain for nitric acid estimation a little mercuric chlorid is placed each month with the view of preventing any change of ammonia into nitric acid. It may be doubted, however, if this precaution is necessary, as the rain water thus collected always contains a very appreciable amount of lead; and experiments have shown that on the whole, rain water more frequently gains than loses ammonia by keeping.
Preparation of the Sample.—The method first employed by Warington was to concentrate ten pounds of the rain water in a retort, a little magnesia being used to decompose any ammonium nitrite or nitrate present. Concentration by evaporation in the open air, and especially over gas, results in a distinct addition to nitrites present. When concentrated to a small bulk, the water is filtered and evaporated to dryness in a very small beaker. The nitrogen as nitrates and nitrites is then determined by means of the methods already described.
DETERMINATION OF FREE AND ALBUMINOID AMMONIA IN RAIN AND DRAINAGE WATERS AND SOIL EXTRACTS.
=511. Nessler Process.=—The quantities of free ammonia in rain and most drainage waters are minute, but may reach considerable magnitude in some sewages. By reason of these minute proportions, gravi- and volumetric methods are not suitable for its quantitive determination. Recourse is therefore had to the delicate colorimetric reaction first proposed by Nessler. This reaction is based on the yellowish-brown coloration produced by ammonia in a solution of mercuric iodid in potassium iodid. The coloration is due to the formation of oxydimercuric ammonium iodid, NH₂Hg₂OI, and takes place between the molecule of free ammonia and the mercuric iodid dissolved in the alkaline potassium iodid as represented by the following equation:
Hg—O—Hg—I Hg / / \ O + 2H₂N = 2O NH₂I + H₂O \ \ / Hg—O—Hg—I HG
Nessler Reagent.—Dissolve thirty-five grams of potassium iodid in 100 cubic centimeters of water. Add to this solution gradually a solution of seventeen grams of mercuric chlorid in 300 cubic centimeters of water until a permanent precipitate of mercuric iodid is formed. Add now enough of a twenty per cent solution of sodium hydroxid to make 1000 cubic centimeters.
The mixed solutions, at room temperature, are treated with additional mercuric chlorid until the precipitate formed, after thorough stirring, remains undissolved. This precipitate is then allowed to subside, and when the supernatant liquid is perfectly clear, it is decanted or filtered through asbestos and kept in a well-stoppered bottle in a dark place. The part in use should be transferred to a smaller bottle as required. The solution should be made for a few days before using, since its delicacy is increased by keeping. The nessler reagent should show a faint yellow tint. If colorless it is not delicate, and shows the addition of an insufficient quantity of mercuric chlorid. When properly prepared, two cubic centimeters of the reagent poured into fifty cubic centimeters of water containing 0.05 milligram of ammonia will at once develop a yellowish-brown tint.
Preparation of Ammonia-Free Water.—To pure distilled water add pure, recently-ignited sodium carbonate, from one to two grams per one liter, and distill. When one-fourth of the whole has passed over, the distillate may be regarded as free from ammonia; fifty cubic centimeters of the following distillate should give no reaction with the nessler reagent. The distillation should be continued until the residual volume in the retort is about one-fourth of the original, and the distillate free of ammonia is carefully preserved in close glass-stoppered bottles previously washed with ammonia-free water. Pure water, free of ammonia may also be obtained by distilling with sulfuric acid.
Comparative Solution of Ammonium Chlorid containing 0.00001 gram Ammonia in one cubic centimeter.—Dissolve 3.15 grams H₄NCl in ammonia-free water and make the volume up to one liter. Take ten cubic centimeters of the above solution and dilute to 1000 with water, free from ammonia.
Solution containing 0.00001 gram Nitrogen in one cubic centimeter.—Dissolve 3.82 grams H₄NCl in water, free from ammonia and dilute with same to 1000 cubic centimeters. Dilute ten cubic centimeters of the above solution to 1000.
The Distillation.—Any kind of suitable retort or flask connected with a good condenser may be used. The capacity of the retort should be from 700 to 1,000 cubic centimeters. The retort and condenser preferred by Leffmann and Beam are shown in Fig. 91. Any good lamp may be used in which the flame is under complete control. The gauze burner shown in the figure is easily controlled and distributes the heat evenly over the surface of the retort thus diminishing the danger of fracture. The apparatus having been previously rinsed with distilled water receives 500 cubic centimeters of the liquid to be tested for ammonia, together with a few pieces of recently ignited pumice stone to prevent bumping and five cubic centimeters of the twenty percent sodium carbonate solution to render its contents alkaline. The water is raised to the boiling-point and with gentle ebullition fifty cubic centimeters of distillate collected. The distillate is conveniently collected in a color-comparison cylinder of thin white glass and flat bottom, about two and a half centimeters in diameter, and marked at fifty and one hundred cubic centimeters. Two cubic centimeters of the nessler reagent are added and if ammonia be present a yellowish-brown color will be developed, the intensity of which is matched by taking portions of the ammonium chlorid solution, diluting to fifty cubic centimeters with pure water and treating with the same quantity of the nessler reagent. The process is repeated until a distillate is obtained which gives no reaction for ammonia. The sum of the quantities obtained in the several distillates gives the total amount of ammonia in the 500 cubic centimeters of the water taken. In most cases practically all the ammonia is obtained in three or four portions of the distillate.
Albuminoid Ammonia.—The residue from the process just described is employed for the purpose of determining the albuminoid ammonia. Two hundred grams of potassium hydroxid and eight grams of potassium permanganate are dissolved in 1,000 parts of distilled water. Fifty cubic centimeters of the solution are placed in a porcelain dish with 100 cubic centimeters of distilled water and evaporated to fifty cubic centimeters. This liquid is placed in the retort and the distillation resumed and continued until an ammonia-free distillate is obtained. The total albuminoid ammonia is determined by taking the sum of the quantities in the several distillates.
=512. Nessler Reagent of Ilosvay.=—To secure greater delicacy in nesslerizing, Ilosvay uses a reagent prepared as follows:[342]
Dissolve two grams of potassium iodid in five cubic centimeters of water, heat the solution gently, and add three grams of mercuric iodid. After the solution is cooled, add an additional portion of three grams of the mercury salt, and then twenty cubic centimeters of water, and wait until the precipitation is complete. After filtering, there are added to the filtrate from twenty to thirty cubic centimeters of a twenty per cent solution of potassium hydroxid. Only the limpid supernatant liquid is used in the analytical work. With this reagent, Ilosvay has been able to detect 0.02 milligram of ammonia in 110 cubic centimeters of water.
AUTHORITIES CITED IN PART SEVENTH.
Footnote 274:
Comptes rendus, Tome 84, pp. 301, et seq. Journal of the Chemical Society, (Transactions), 1878, p. 44; 1879, p. 429; 1884, p. 637. American Chemical Journal, Vol. 4, p. 452. Proceedings of the American Association for the Advancement of Science, Vol. 41, p. 105. Annales de l’Institut Pasteur, Tome 4, pp. 218, 257, 760; Tome 5, p. 92.
Footnote 275:
Comptes rendus, Tome 118, p. 604.
Footnote 276:
Bulletin de la Academie royale de Belgique, [3], Tome 25, p. 727. Journal of the Chemical Society, (Abstracts), June, 1894, p. 248.
Footnote 277:
Chemical News, Oct. 13, 1893, p. 176.
Footnote 278:
Comptes rendus, Tome 109, p. 883.
Footnote 279:
Op. cit. supra, Tome 89, pp. 891, et seq.
Footnote 280:
Journal of the Chemical Society, (Transactions), Vol. 45, pp. 645, et seq.
Footnote 281:
Jahresbericht der Agricultur Chemie, 1881, S. 43.
Footnote 282:
Annual Report of the British Board of Health, 1883.
Footnote 283:
Annales de l’Institut Pasteur, 1891, S. 93.
Footnote 284:
Philosophical Transactions of the Royal Society of London, Vol. 181, (1890).
Footnote 285:
Zeitschrift für Biologie, Band 9, S. 172.
Footnote 286:
Archives de Science Biologique à St. Petersbourgh, Tome 1, p. 1331.
Footnote 287:
Annales de l’Institut Pasteur, 1891, pp. 581, et seq.
Footnote 288:
Op. cit. supra, 1891, Plate 18, Fig. 2.
Footnote 289:
Op. cit. supra, 1891, pp. 595, et seq.
Footnote 290:
Op. cit. supra, 1891, Plate 18, Fig. 1.
Footnote 291:
Journal of the Chemical Society, (Transactions), 1891, pp. 498, et seq.
Footnote 292:
Annales de l’Institut Pasteur, 1891, pp. 605, et seq.
Footnote 293:
Journal of the Chemical Society, (Transactions), 1882, p. 357.
Footnote 294:
Annales de Chimie et de Physique, 1854, Tome 40, p. 479. Zeitschrift für analytische Chemie, 1870, S. 24; 1877, S. 291. Die Landwirtschaftlichen Versuchs-Stationen, Band 12, S. 164. Journal of the Chemical Society, (Transactions), 1880, p. 468; 1882, p. 345; 1889, p. 537.
Footnote 295:
Encyclopedie Chimique, Tome 4, p. 151.
Footnote 296:
Annales de la Science Agronomique, 1891, pp. 263, et seq.
Footnote 297:
Berichte der deutschen chemischen Gesellschaft, Band 23, S. 1361.
Footnote 298:
Zeitschrift für analytische Chemie, Band 9, S. 24, 401. Die Landwirtschaftlichen Versuchs-Stationen, Band 9, S. 9. Berichte der deutschen chemischen Gesellschaft, Band 6, S. 1038.
Footnote 299:
Zeitschrift für analytische Chemie, Band 33, S. 200.
Footnote 300:
Apotheker Zeitung, 1891, Band 5, S. 287.
Footnote 301:
Sutton’s Volumetric Analysis, 3d edition, p. 316. Warington, Journal of the Chemical Society, (Transactions), 1879, p. 376.
Footnote 302:
Report of the National Board of Health, 1882, p. 281.
Footnote 303:
Berichte der deutschen chemischen Gesellschaft, Band 11, S. 432.
Footnote 304:
Bulletin de la Société Chimique, [3], Tomes 11–12, p. 625.
Footnote 305:
Encyclopedie Chimique, Tome 4, p. 154.
Footnote 306:
Zeitschrift für analytische Chemie, Band 7, S. 412. Fresenius, Quantitative Analysis, Grove’s translation, special part, p. 118.
Footnote 307:
Journal of the Chemical Society, (Transactions), 1879, pp. 578, et seq.
Footnote 308:
Bulletin 38, Department of Agriculture, Division of Chemistry, p. 204.
Footnote 309:
Die Landwirtschaftlichen Versuchs-Stationen, Band 41, S. 165.
Footnote 310:
Chemiker Zeitung, 1892, Band 16, S. 1952.
Footnote 311:
Zeitschrift für angewandte Chemie, 1893, S. 161.
Footnote 312:
Chemiker Zeitung, 1889, No. 15.
Footnote 313:
Vid. op. cit. 38, 1890, S. 695.
Footnote 314:
Archives de la Société Physique de Genève, Tome 31, p. 352.
Footnote 315:
Chemiker Zeitung, 1890, S. 1410.
Footnote 316:
Chemisches Centralblatt, 1890, Band 2, S. 926.
Footnote 317:
Vid. op. cit. 38, 1891, S. 241.
Footnote 318:
Vid. op. cit. 34, 1889, p. 538.
Footnote 319:
Op. cit. supra, 1881, p. 100.
Footnote 320:
Op. cit. supra, Vol. 57, p. 811.
Footnote 321:
Op. cit. supra, 1891, pp. 530, et seq.
Footnote 322:
Op. cit. supra, 1874, p. 630, and 1885, p. 86.
Footnote 323:
Sutton’s Volumetric Analysis, 4th edition, p. 103.
Footnote 324:
American Journal of Science, Vol. 44, p. 117.
Footnote 325:
American Chemical Journal, Vol. 11, p. 249.
Footnote 326:
Journal of the Franklin Institute, Vol. 127, p. 61.
Footnote 327:
Zeitschrift für Hygiene, Band 2, S. 163.
Footnote 328:
Chemical News, 1889, Nov. 29, 261.
Footnote 329:
Vid. op. cit. supra, p. 51.
Footnote 330:
Examination of Water for Sanitary and Technical Purposes, p. 28.
Footnote 331:
Chemical News, 1890, Jan. 10, p. 15.
Footnote 332:
Zeitschrift für angewandte Chemie, 1894, Heft 12, S. 347.
Footnote 333:
Journal of the Chemical Society, (Abstracts), 1891, p. 489.
Footnote 334:
Zeitschrift für analytische Chemie, Band 18, S. 597. Zeitschrift für angewandte Chemie, 1889, S. 666. Bulletin de la Société Chimique, [3], Tome 2, p. 347.
Footnote 335:
Op. cit. 57, p. 30.
Footnote 336:
Zeitschrift für angewandte Chemie, 1894, S. 349.
Footnote 337:
Bulletin de la Société Chimique, [3], Tomes 11–12, p. 218.
Footnote 338:
Encyclopedie Chimique, Tome 4, p. 262.
Footnote 339:
Peligot, Traité de Chimie Analytique appliqueè à Agriculture, p. 261.
Footnote 340:
Berichte der deutschen chemischen Gesellschaft, 1893, S. 589.
Footnote 341:
Journal of the Chemical Society, 1889, p. 537.
Footnote 342:
Op. cit. 64, p. 216.
NOTE.—On page 158, paragraph 172, third line, insert, “and determining matters dissolved therein,” after “flow.”